Cell-free protein synthesis system, reaction mixture used therefor and method for producing protein

Abstract

Disclosed herein are cell-free protein synthesis systems, reaction mixtures for use in the systems, and methods for preparing proteins of interest.

Description

Background Art and Summary of the Invention

[0001] Cell-free protein synthesis (CFPS) offers many advantages over cell-based protein synthesis, such as time savings and increased total yields of functional soluble full-length proteins. Additionally, CFPS is less sensitive to toxic proteins and is suitable for high-throughput experiments.

[0002] Commercial CFPS mixtures typically require complex and expensive raw materials, such as free amino acids, free ribonucleotides, and free NADH / NADPH. Although commercial CFPS typically emphasizes reaction speed, its yield is often limited because the raw materials are the rate-limiting step, as the raw materials must be produced and regenerated before they can be converted into energy and / or protein products. Additionally, commercial CFPS typically relies on systems that need to be stored at lower temperatures (e.g., -20°C).

[0003] In contrast, the present application discloses novel CFPS systems that contain their novel reaction mixtures, which use simpler and cheaper raw materials and avoid many of the problems faced by commercial CFPS (e.g., wasteful consumption of amino acids in side reactions, regeneration of NADH / NADPH) and provide the benefit of cost reduction. Additionally, these novel CFPS systems and these reaction mixtures can be stored at 4°C and / or room temperature, thereby avoiding the inconveniences and costs associated with storage at lower temperatures. Brief Description of the Drawings

[0004] The novel features of the present invention are specifically set forth in the appended claims. The features and advantages of the present invention will be better understood by reference to the following detailed description and the drawings, which set forth illustrative embodiments that utilize the principles of the present invention, in which:

[0005] Figure 1 Shows the effects of extract and mixture conditions and temperature on the yield of CFPS deGFP protein. "Min" and "Com" refer to "minimal" and "commercial", respectively, and "CE" refers to cell extract.

[0006] Figure 2 Shows the effects of removing individual reaction mixture components on the yield of CFPS deGFP protein when using minimal cell extract.

[0007] Figure 3 Shows the effects of sodium chloride concentration on the yield of CFPS protein when using minimal cell extract from Vibrio natriegens (V. natriegens). Detailed Description Definitions

[0008] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. It should be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and do not limit any claimed subject matter. If any material incorporated by reference into this disclosure is inconsistent with the explicit content of this disclosure, the explicit content shall prevail. In this application, unless otherwise explicitly stated, the use of the singular includes the plural. It must be noted that, unless the context clearly dictates otherwise, as used in this specification and the appended claims, the singular forms “a / an” and “the” include plural referents. In this application, unless otherwise stated, the use of “or” means “and / or”. Additionally, the use of the term “including” and other forms such as “include”, “includes” and “included” is not limiting.

[0009] References in this specification to “some embodiments”, “an embodiment”, “one embodiment” or “other embodiments” mean that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments of the invention, but not necessarily all embodiments.

[0010] As used herein, ranges and amounts can be expressed as “about” a particular value or range. “About” also includes the exact amount. Thus, “about 5 μL” means “about 5 μL” as well as “5 μL”. Generally, the term “about” includes amounts that are expected to be within experimental error. For example, the term

[0011] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0012] As used herein, a “cell-free” or “CF” system or its elements are systems or elements that perform their intended function (e.g., protein synthesis) by primarily utilizing the non-living cell aspects of the system or element (e.g., the supernatant collected from lysed bacteria). A “cell-free” system or its elements can contain residual amounts of living cells.

[0013] As used herein, "minimal medium" is a culture medium that contains only a minimal set of nutrients required for bacterial growth. The nutrients can vary depending on the bacterial species and will be known to the person skilled in the art for a given species. In an embodiment, the "minimal medium" consists essentially of: (a) at least one carbon source (e.g., a sugar such as glucose, a polyol such as glycerol, or a dicarboxylic acid such as succinate), (b) at least one of an ammonium source (e.g., ammonium sulfate), a phosphate source (e.g., potassium dihydrogen phosphate or dipotassium hydrogen phosphate), a sulfur source, a magnesium source (e.g., magnesium sulfate), and a sodium salt (e.g., sodium succinate), and (c) water. The "minimal medium" can contain elements for structural support for bacterial culture but not used as a nutrient source for the bacteria (e.g., agar). Preferably, the "minimal medium" does not contain vitamins or coenzymes. Preferably, the "minimal medium" does not contain amino acids or proteins.

[0014] As used herein, "DNA" or "RNA" encoding a protein of interest refers to one or more deoxyribonucleic acids or ribonucleic acids having at least a portion of the codons or anticodons encoding the protein of interest. The protein of interest can be encoded by more than one deoxyribonucleic acid or ribonucleic acid or a combination thereof. The DNA or RNA can be circular or linear, and the DNA can be single-stranded or double-stranded. In some embodiments, the DNA or RNA can be a plasmid.

[0015] As used herein, "free amino acid" refers to an amino acid that is not part of a polypeptide or protein.

[0016] As used herein, "free nucleotide" refers to a nucleotide that is not part of a polynucleotide and encompasses the corresponding nucleoside. Exemplary polynucleotides include, but are not limited to, plasmids, genomic DNA, and genomic RNA not covered by the "free nucleotide". A "nucleotide" refers to a compound containing a nucleoside moiety and a phosphate moiety. Exemplary nucleotides include, but are not limited to, adenosine triphosphate (ATP), uridine triphosphate (UTP), cytidine triphosphate (CTP), guanosine triphosphate (GTP), adenosine diphosphate (ADP), uridine diphosphate (UDP), cytidine diphosphate (CDP), guanosine diphosphate (GDP), adenosine monophosphate (AMP), uridine monophosphate (UMP), cytidine monophosphate (CMP), and guanosine monophosphate (GMP), deoxyadenosine triphosphate (dATP), deoxythymidine triphosphate (dTTP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxyadenosine diphosphate (dADP), thymidine diphosphate (dTDP), deoxycytidine diphosphate (dCDP), deoxyguanosine diphosphate (dGDP), deoxyadenosine monophosphate (dAMP), deoxythymidine monophosphate (dTMP), deoxycytidine monophosphate (dCMP), and deoxyguanosine monophosphate (dGMP).

[0017] As used herein, "free NADH / NADPH" refers to any combination and ratio of free oxidized and reduced forms of nicotinamide adenine dinucleotide and free oxidized and reduced forms of nicotinamide adenine dinucleotide phosphate.

[0018] As used herein, "maintaining" a cell-free protein synthesis system at a certain temperature means keeping the cell-free protein synthesis system in a space (e.g., a room) where its temperature is that certain temperature for a time period sufficient to perform cell-free protein synthesis. One or more parts of the cell-free protein synthesis system may have a temperature that is higher or lower than that certain temperature of the space.

[0019] Although various features of the invention may be described in the context of a single embodiment, these features may also be provided separately or in any suitable combination. Conversely, although the invention may be described herein in the context of separate embodiments for clarity, the invention may also be practiced in a single embodiment. Cell-free protein synthesis system

[0020] The inventors have developed a cell-free protein synthesis system that comprises a cell extract from bacteria grown in minimal medium and does not require the addition of amino acids or nucleotides. It is thought to operate by generating these components via the use of the aerobic respiration cycle.

[0021] Accordingly, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria.

[0022] In some embodiments, the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0. In some embodiments, the pH of the cell-free protein synthesis system is from 7.1 to 7.9. In some embodiments, the pH of the cell-free protein synthesis system is from 7.2 to 7.8. In some embodiments, the pH of the cell-free protein synthesis system is from 7.3 to 7.7. In some embodiments, the pH of the cell-free protein synthesis system is from 7.4 to 7.6. In some embodiments, the pH of the cell-free protein synthesis system is about 7.5. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, and wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, from 7.1 to 7.9, from 7.2 to 7.8, from 7.3 to 7.7, from 7.4 to 7.6, or about 7.5.

[0023] In some embodiments, the cell-free protein synthesis system is maintained at a temperature below about 30 °C. In some embodiments, the cell-free protein synthesis system is maintained at a temperature from about 20 °C to about 30 °C. In some embodiments, the cell-free protein synthesis system is maintained at a temperature from about 21 °C to about 29 °C. In some embodiments, the cell-free protein synthesis system is maintained at a temperature from about 22 °C to about 28 °C. In some embodiments, the cell-free protein synthesis system is maintained at a temperature from about 23 °C to about 27 °C. In some embodiments, the cell-free protein synthesis system is maintained at a temperature from about 24 °C to about 36 °C. In some embodiments, the cell-free protein synthesis system is maintained at a temperature above -20 °C. In some embodiments, the cell-free protein synthesis system is maintained at a temperature of about 25 °C. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, and wherein the cell-free protein synthesis system is maintained at a temperature below about 30 °C.

[0024] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, and wherein the cell-free protein synthesis system is maintained at a temperature below about 30°C.

[0025] In some embodiments, the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer.

[0026] In some embodiments, the reaction mixture further comprises at least two of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer.

[0027] In some embodiments, the reaction mixture further comprises at least three of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer.

[0028] In some embodiments, the reaction mixture further comprises at least four of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer.

[0029] In some embodiments, the reaction mixture further comprises at least five of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer.

[0030] In some embodiments, the reaction mixture further comprises at least six of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer.

[0031] In some embodiments, the reaction mixture further comprises at least seven of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer.

[0032] In some embodiments, the reaction mixture further comprises: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and optionally (h) a buffer.

[0033] In some embodiments, the reaction mixture further comprises: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer.

[0034] In some embodiments, the reaction mixture contains NaCl. The NaCl can be present at 50 - 250 mM, such as 100 - 200 mM, such as 100 mM. Such NaCl concentrations are advantageous in cases where cell extracts from Vibrio natriegens are used.

[0035] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless the free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, and wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer. In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless the free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the cell-free protein synthesis system is maintained at a temperature below about 30 °C, and wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer.

[0036] In some embodiments, the molecular crowding agent is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin. In some embodiments, the molecular crowding agent comprises PEG 400. In some embodiments, the molecular crowding agent comprises PEG 1500. In some embodiments, the molecular crowding agent comprises PEG3350. In some embodiments, the molecular crowding agent comprises PEG 4000. In some embodiments, the molecular crowding agent comprises PEG 6000. In some embodiments, the molecular crowding agent comprises PEG 8000. In some embodiments, the molecular crowding agent comprises maltodextrin. In some embodiments, the molecular crowding agent comprises Ficoll 70. In some embodiments, the molecular crowding agent comprises Ficoll 400. In some embodiments, the molecular crowding agent comprises dextran. In some embodiments, the molecular crowding agent comprises serum albumin. In some embodiments, the molecular crowding agent comprises bovine serum albumin. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, and wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowding agent; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; (g) a sulfur source; and (h) a buffer, and wherein the molecular crowding agent (if present) is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin.

[0037] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the cell-free protein synthesis system is maintained at a temperature below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowding agent; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, and wherein the molecular crowding agent (if present) is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin.

[0038] In some embodiments, the concentration of the molecular crowder in the cell-free protein synthesis system is from about 0 to 40 g / L. In some embodiments, the concentration of the molecular crowder in the cell-free protein synthesis system is from 2.5 to 37.5 g / L. In some embodiments, the concentration of the molecular crowder in the cell-free protein synthesis system is from 5 to 35 g / L. In some embodiments, the concentration of the molecular crowder in the cell-free protein synthesis system is from 7.5 to 32.5 g / L. In some embodiments, the concentration of the molecular crowder in the cell-free protein synthesis system is from 10 to 30 g / L. In some embodiments, the concentration of the molecular crowder in the cell-free protein synthesis system is from 12.5 to 27.5 g / L. In some embodiments, the concentration of the molecular crowder in the cell-free protein synthesis system is from 15 to 25 g / L. In some embodiments, the concentration of the molecular crowder in the cell-free protein synthesis system is from 17.5 to 22.5 g / L. In some embodiments, the concentration of the molecular crowder in the cell-free protein synthesis system is 20 g / L. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, and wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, and wherein the molecular crowder (if present) is at a concentration in the cell-free protein synthesis system of from about 0 to 40 g / L.

[0039] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless such free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the cell-free protein synthesis system is maintained at a temperature below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder (if present) is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, and wherein the molecular crowder (if present) is present in the cell-free protein synthesis system at a concentration of from about 0 to 40 g / L.

[0040] In some embodiments, the carbon source includes sugars. In some embodiments, the carbon source is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose. In some embodiments, the carbon source includes glucose. In some embodiments, the carbon source includes fructose. In some embodiments, the carbon source includes galactose. In some embodiments, the carbon source includes sucrose. In some embodiments, the carbon source includes lactose. In some embodiments, the carbon source includes gluconate. In some embodiments, the carbon source includes starch. In some embodiments, the carbon source includes maltodextrin. In some embodiments, the carbon source includes maltose. In some embodiments, alternatively, the carbon source does not include maltose. In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowding agent; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, and wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose.

[0041] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the cell-free protein synthesis system is maintained at a temperature below about 30 °C, wherein the molecular crowding agent (if present) is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowding agent in the cell-free protein synthesis system is from about 0 to 40 g / L, and wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose.

[0042] In some embodiments, the concentration of the carbon source in the cell-free protein synthesis system is from about 0 to 50 g / L. In some embodiments, the concentration of the carbon source in the cell-free protein synthesis system is from 2 to 47 g / L. In some embodiments, the concentration of the carbon source in the cell-free protein synthesis system is from 4 to 44 g / L. In some embodiments, the concentration of the carbon source in the cell-free protein synthesis system is from 6 to 41 g / L. In some embodiments, the concentration of the carbon source in the cell-free protein synthesis system is from 8 to 38 g / L. In some embodiments, the concentration of the carbon source in the cell-free protein synthesis system is from 10 to 35 g / L. In some embodiments, the concentration of the carbon source in the cell-free protein synthesis system is from 12 to 32 g / L. In some embodiments, the concentration of the carbon source in the cell-free protein synthesis system is from 14 to 29 g / L. In some embodiments, the concentration of the carbon source in the cell-free protein synthesis system is from 16 to 36 g / L. In some embodiments, the concentration of the carbon source in the cell-free protein synthesis system is from 18 to 33 g / L. In some embodiments, the concentration of the carbon source in the cell-free protein synthesis system is 20 g / L. In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, and wherein the carbon source (if present) in the cell-free protein synthesis system is at a concentration from about 0 to 50 g / L.

[0043] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the cell-free protein synthesis system is maintained at a temperature below about 30°C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder (if present) is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowder (if present) in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, and wherein the concentration of the carbon source in the cell-free protein synthesis system is from about 0 to 50 g / L.

[0044] In some embodiments, the ammonium source includes ammonium sulfate. In some embodiments, the concentration of the ammonium source in the cell-free protein synthesis system is from 2 to 20 mM. In some embodiments, the concentration of the ammonium source in the cell-free protein synthesis system is from 3 to 19 mM. In some embodiments, the concentration of the ammonium source in the cell-free protein synthesis system is from 4 to 18 mM. In some embodiments, the concentration of the ammonium source in the cell-free protein synthesis system is from 5 to 17 mM. In some embodiments, the concentration of the ammonium source in the cell-free protein synthesis system is from 6 to 16 mM. In some embodiments, the concentration of the ammonium source in the cell-free protein synthesis system is from 7 to 16 mM. In some embodiments, the concentration of the ammonium source in the cell-free protein synthesis system is from 8 to 16 mM. In some embodiments, the concentration of the ammonium source in the cell-free protein synthesis system is from 9 to 15 mM. In some embodiments, the concentration of the ammonium source in the cell-free protein synthesis system is from 10 to 14 mM. In some embodiments, the concentration of the ammonium source in the cell-free protein synthesis system is from 11 to 13 mM. In some embodiments, the concentration of the ammonium source in the cell-free protein synthesis system is 12 mM. In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless the free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, and wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowding agent; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, and wherein the ammonium source includes ammonium sulfate.In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, and wherein the ammonium source (if present) is at a concentration of from 2 to 20 mM in the cell-free protein synthesis system.

[0045] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the cell-free protein synthesis system is maintained at a temperature below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder (if present) is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the molecular crowder (if present) is at a concentration of from about 0 to 40 g / L in the cell-free protein synthesis system, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the carbon source (if present) is at a concentration of from about 0 to 50 g / L in the cell-free protein synthesis system, wherein the ammonium source (if present) comprises ammonium sulfate, and wherein the ammonium source is at a concentration of from 2 to 20 mM in the cell-free protein synthesis system.

[0046] In some embodiments, the potassium source is selected from potassium glutamate and potassium gluconate. In some embodiments, the potassium source comprises potassium glutamate and potassium gluconate. In some embodiments, the potassium source comprises potassium glutamate. In some embodiments, the potassium source comprises potassium gluconate. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, and wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, and wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate.

[0047] In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in minimal media; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the cell-free protein synthesis system is maintained at a temperature below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder (if present) is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the molecular crowder (if present) is present in the cell-free protein synthesis system at a concentration of from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the carbon source (if present) is present in the cell-free protein synthesis system at a concentration of from about 0 to 50 g / L, wherein the ammonium source (if present) comprises ammonium sulfate, wherein the ammonium source (if present) is present in the cell-free protein synthesis system at a concentration of from 2 to 20 mM, and wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate.

[0048] In some embodiments, the concentration of potassium glutamate in the cell-free protein synthesis system ranges from about 10 to 270 mM. In some embodiments, the concentration of potassium glutamate in the cell-free protein synthesis system ranges from 11 to 240 mM. In some embodiments, the concentration of potassium glutamate in the cell-free protein synthesis system ranges from 12 to 210 mM. In some embodiments, the concentration of potassium glutamate in the cell-free protein synthesis system ranges from 13 to 180 mM. In some embodiments, the concentration of potassium glutamate in the cell-free protein synthesis system ranges from 14 to 150 mM. In some embodiments, the concentration of potassium glutamate in the cell-free protein synthesis system ranges from 15 to 120 mM. In some embodiments, the concentration of potassium glutamate in the cell-free protein synthesis system ranges from 16 to 90 mM. In some embodiments, the concentration of potassium glutamate in the cell-free protein synthesis system ranges from 17 to 60 mM. In some embodiments, the concentration of potassium glutamate in the cell-free protein synthesis system ranges from 18 to 30 mM. In some embodiments, the concentration of potassium glutamate in the cell-free protein synthesis system ranges from 18 to 28 mM. In some embodiments, the concentration of potassium glutamate in the cell-free protein synthesis system ranges from 18 to 26 mM. In some embodiments, the concentration of potassium glutamate in the cell-free protein synthesis system ranges from 18 to 24 mM. In some embodiments, the concentration of potassium glutamate in the cell-free protein synthesis system ranges from 19 to 24 mM. In some embodiments, the concentration of potassium glutamate in the cell-free protein synthesis system ranges from 19 to 22 mM. In some embodiments, the concentration of potassium glutamate in the cell-free protein synthesis system ranges from 19 to 21 mM. In some embodiments, the concentration of potassium glutamate in the cell-free protein synthesis system is 20 mM. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, and wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowding agent; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, and wherein the concentration of potassium glutamate (if present) in the cell-free protein synthesis system ranges from about 10 to 270 mM.

[0049] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the cell-free protein synthesis system is maintained at a temperature below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; (g) a sulfur source; and (h) a buffer, wherein the molecular crowder (if present) is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the molecular crowder (if present) is present in the cell-free protein synthesis system at a concentration of from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the carbon source (if present) is present in the cell-free protein synthesis system at a concentration of from about 0 to 50 g / L, wherein the ammonium source (if present) comprises ammonium sulfate, wherein the ammonium source (if present) is present in the cell-free protein synthesis system at a concentration of from 2 to 20 mM, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, and wherein the potassium glutamate (if present) is present in the cell-free protein synthesis system at a concentration of from about 10 to 270 mM.

[0050] In some embodiments, the concentration of potassium gluconate in the cell-free protein synthesis system is from about 10 to 250 mM. In some embodiments, the concentration of potassium gluconate in the cell-free protein synthesis system is from 15 to 240 mM. In some embodiments, the concentration of potassium gluconate in the cell-free protein synthesis system is from 20 to 230 mM. In some embodiments, the concentration of potassium gluconate in the cell-free protein synthesis system is from 25 to 220 mM. In some embodiments, the concentration of potassium gluconate in the cell-free protein synthesis system is from 30 to 210 mM. In some embodiments, the concentration of potassium gluconate in the cell-free protein synthesis system is from 35 to 200 mM. In some embodiments, the concentration of potassium gluconate in the cell-free protein synthesis system is from 40 to 190 mM. In some embodiments, the concentration of potassium gluconate in the cell-free protein synthesis system is from 45 to 180 mM. In some embodiments, the concentration of potassium gluconate in the cell-free protein synthesis system is from 50 to 170 mM. In some embodiments, the concentration of potassium gluconate in the cell-free protein synthesis system is from 55 to 160 mM. In some embodiments, the concentration of potassium gluconate in the cell-free protein synthesis system is from 60 to 150 mM. In some embodiments, the concentration of potassium gluconate in the cell-free protein synthesis system is from 70 to 140 mM. In some embodiments, the concentration of potassium gluconate in the cell-free protein synthesis system is from 80 to 130 mM. In some embodiments, the concentration of potassium gluconate in the cell-free protein synthesis system is from 90 to 120 mM. In some embodiments, the concentration of potassium gluconate in the cell-free protein synthesis system is from 95 to 110 mM. In some embodiments, the concentration of potassium gluconate in the cell-free protein synthesis system is from 95 to 105 mM. In some embodiments, the concentration of potassium gluconate in the cell-free protein synthesis system is 100 mM.In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, and wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowding agent; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, and wherein the potassium gluconate (if present) is present in the cell-free protein synthesis system at a concentration from about 10 to 250 mM.

[0051] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the cell-free protein synthesis system is maintained at a temperature below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; (g) a sulfur source; and (h) a buffer, wherein the molecular crowder (if present) is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the molecular crowder (if present) is present in the cell-free protein synthesis system at a concentration of from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the carbon source (if present) is present in the cell-free protein synthesis system at a concentration of from about 0 to 50 g / L, wherein the ammonium source (if present) comprises ammonium sulfate, wherein the ammonium source (if present) is present in the cell-free protein synthesis system at a concentration of from 2 to 20 mM, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, wherein the potassium glutamate (if present) is present in the cell-free protein synthesis system at a concentration of from about 10 to 270 mM, and wherein the potassium gluconate (if present) is present in the cell-free protein synthesis system at a concentration of from about 10 to 250 mM.

[0052] In some embodiments, the magnesium source includes magnesium glutamate. In some embodiments, the concentration of the magnesium source in the cell-free protein synthesis system ranges from 0 to about 10 mM. In some embodiments, the concentration of the magnesium source in the cell-free protein synthesis system ranges from 0.5 to 9.25 mM. In some embodiments, the concentration of the magnesium source in the cell-free protein synthesis system ranges from 1 to 8.5 mM. In some embodiments, the concentration of the magnesium source in the cell-free protein synthesis system ranges from 1.5 to 7.75 mM. In some embodiments, the concentration of the magnesium source in the cell-free protein synthesis system ranges from 2 to 7 mM. In some embodiments, the concentration of the magnesium source in the cell-free protein synthesis system ranges from 2.5 to 6.25 mM. In some embodiments, the concentration of the magnesium source in the cell-free protein synthesis system ranges from 3 to 5.5 mM. In some embodiments, the concentration of the magnesium source in the cell-free protein synthesis system ranges from 3.5 to 4.75 mM. In some embodiments, the concentration of the magnesium source in the cell-free protein synthesis system ranges from 3.5 to 4.5 mM. In some embodiments, the concentration of the magnesium source in the cell-free protein synthesis system ranges from 3.75 to 4.25 mM. In some embodiments, the concentration of the magnesium source in the cell-free protein synthesis system is 4 mM. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, and wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, and wherein the magnesium source (if present) includes magnesium glutamate.In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, and wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the magnesium source (if present) comprises magnesium glutamate, and wherein the concentration of the magnesium source (if present) in the cell-free protein synthesis system is from 0 to about 10 mM.

[0053] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the cell-free protein synthesis system is maintained at a temperature below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder (if present) is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the molecular crowder (if present) is present in the cell-free protein synthesis system at a concentration of from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the carbon source (if present) is present in the cell-free protein synthesis system at a concentration of from about 0 to 50 g / L, wherein the ammonium source (if present) comprises ammonium sulfate, wherein the ammonium source (if present) is present in the cell-free protein synthesis system at a concentration of from 2 to 20 mM, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, wherein the potassium glutamate (if present) is present in the cell-free protein synthesis system at a concentration of from about 10 to 270 mM, wherein the potassium gluconate (if present) is present in the cell-free protein synthesis system at a concentration of from about 10 to 250 mM, wherein the magnesium source (if present) comprises magnesium glutamate, and wherein the magnesium source (if present) is present in the cell-free protein synthesis system at a concentration of from 0 to about 10 mM.

[0054] In some embodiments, the phosphate source comprises potassium phosphate. In some embodiments, the concentration of the phosphate source in the cell-free protein synthesis system is from about 5 to about 23.5 mM. In some embodiments, the concentration of the phosphate source in the cell-free protein synthesis system is from 6 to 22 mM. In some embodiments, the concentration of the phosphate source in the cell-free protein synthesis system is from 7 to 20.5 mM. In some embodiments, the concentration of the phosphate source in the cell-free protein synthesis system is from 8 to 19 mM. In some embodiments, the concentration of the phosphate source in the cell-free protein synthesis system is from 9 to 17.5 mM. In some embodiments, the concentration of the phosphate source in the cell-free protein synthesis system is from 10 to 16 mM. In some embodiments, the concentration of the phosphate source in the cell-free protein synthesis system is from 11 to 14.5 mM. In some embodiments, the concentration of the phosphate source in the cell-free protein synthesis system is from 12 to 14 mM. In some embodiments, the concentration of the phosphate source in the cell-free protein synthesis system is from 12.5 to 13.5 mM. In some embodiments, the concentration of the phosphate source in the cell-free protein synthesis system is 13 mM. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, and wherein the phosphate source comprises potassium phosphate.In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, and wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; (g) a sulfur source; and (h) a buffer, and wherein the concentration of the phosphate source in the cell-free protein synthesis system is from about 5 to about 23.5 mM.

[0055] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the cell-free protein synthesis system is maintained at a temperature below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowding agent; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowding agent is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowding agent (if present) in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the concentration of the carbon source (if present) in the cell-free protein synthesis system is from about 0 to 50 g / L, wherein the ammonium source (if present) comprises ammonium sulfate, wherein the concentration of the ammonium source (if present) in the cell-free protein synthesis system is from 2 to 20 mM, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, wherein the concentration of potassium glutamate (if present) in the cell-free protein synthesis system is from about 10 to 270 mM, wherein the concentration of potassium gluconate (if present) in the cell-free protein synthesis system is from about 10 to 250 mM, wherein the magnesium source (if present) comprises magnesium glutamate, wherein the concentration of the magnesium source (if present) in the cell-free protein synthesis system is from 0 to about 10 mM, wherein the phosphate source (if present) comprises potassium phosphate, and wherein the concentration of the phosphate source (if present) in the cell-free protein synthesis system is from about 5 to about 23.5 mM.

[0056] In some embodiments, the sulfur source comprises ammonium sulfate. In some embodiments, the concentration of the sulfur source in the cell-free protein synthesis system is from 2 to 20 mM. In some embodiments, the concentration of the sulfur source in the cell-free protein synthesis system is from 3 to 19 mM. In some embodiments, the concentration of the sulfur source in the cell-free protein synthesis system is from 4 to 18 mM. In some embodiments, the concentration of the sulfur source in the cell-free protein synthesis system is from 5 to 17 mM. In some embodiments, the concentration of the sulfur source in the cell-free protein synthesis system is from 6 to 16 mM. In some embodiments, the concentration of the sulfur source in the cell-free protein synthesis system is from 7 to 15 mM. In some embodiments, the concentration of the sulfur source in the cell-free protein synthesis system is from 8 to 15 mM. In some embodiments, the concentration of the sulfur source in the cell-free protein synthesis system is from 9 to 14 mM. In some embodiments, the concentration of the sulfur source in the cell-free protein synthesis system is from 10 to 14 mM. In some embodiments, the concentration of the sulfur source in the cell-free protein synthesis system is from 11 to 13 mM. In some embodiments, the concentration of the sulfur source in the cell-free protein synthesis system is from 11.5 to 12.5 mM. In some embodiments, the concentration of the sulfur source in the cell-free protein synthesis system is 12 mM. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, and wherein the sulfur source comprises ammonium sulfate.In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, and wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowding agent; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, and wherein the sulfur source, if present, is at a concentration in the cell-free protein synthesis system of from 2 to 20 mM.

[0057] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the cell-free protein synthesis system is maintained at a temperature below about 30°C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder (if present) is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowder (if present) in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the concentration of the carbon source (if present) in the cell-free protein synthesis system is from about 0 to 50 g / L, wherein the ammonium source (if present) comprises ammonium sulfate, wherein the concentration of the ammonium source (if present) in the cell-free protein synthesis system is from 2 to 20 mM, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, wherein the concentration of potassium glutamate (if present) in the cell-free protein synthesis system is from about 10 to 270 mM, wherein the concentration of potassium gluconate (if present) in the cell-free protein synthesis system is from about 10 to 250 mM, wherein the magnesium source (if present) comprises magnesium glutamate, wherein the concentration of the magnesium source (if present) in the cell-free protein synthesis system is from 0 to about 10 mM, wherein the phosphate source (if present) comprises potassium phosphate, wherein the concentration of the phosphate source (if present) in the cell-free protein synthesis system is from about 5 to about 23.5 mM, wherein the sulfur source (if present) comprises ammonium sulfate, and wherein the concentration of the sulfur source (if present) in the cell-free protein synthesis system is from 2 to 20 mM.

[0058] In some embodiments, the cell-free protein synthesis system comprises a buffer. In some embodiments, the buffer comprises 3-(N-morpholino)propanesulfonic acid (MOPS) buffer. In some embodiments, the concentration of the buffer in the cell-free protein synthesis system ranges from 50 to 300 mM. In some embodiments, the concentration of the buffer in the cell-free protein synthesis system ranges from 55 to 280 mM. In some embodiments, the concentration of the buffer in the cell-free protein synthesis system ranges from 60 to 260 mM. In some embodiments, the concentration of the buffer in the cell-free protein synthesis system ranges from 65 to 240 mM. In some embodiments, the concentration of the buffer in the cell-free protein synthesis system ranges from 70 to 220 mM. In some embodiments, the concentration of the buffer in the cell-free protein synthesis system ranges from 75 to 200 mM. In some embodiments, the concentration of the buffer in the cell-free protein synthesis system ranges from 80 to 180 mM. In some embodiments, the concentration of the buffer in the cell-free protein synthesis system ranges from 85 to 160 mM. In some embodiments, the concentration of the buffer in the cell-free protein synthesis system ranges from 90 to 140 mM. In some embodiments, the concentration of the buffer in the cell-free protein synthesis system ranges from 95 to 120 mM. In some embodiments, the concentration of the buffer in the cell-free protein synthesis system ranges from 95 to 110 mM. In some embodiments, the concentration of the buffer in the cell-free protein synthesis system ranges from 95 to 105 mM. In some embodiments, the concentration of the buffer in the cell-free protein synthesis system is 100 mM. In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowding agent; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, and wherein the buffer (if present) comprises 3-(N-morpholino)propanesulfonic acid (MOPS) buffer.In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, and wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowding agent; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, and wherein the buffer (if present) is present in the cell-free protein synthesis system at a concentration of from 50 to 300 mM.

[0059] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the cell-free protein synthesis system is maintained at a temperature below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder (if present) is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the molecular crowder (if present) is present in the cell-free protein synthesis system at a concentration of from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the carbon source (if present) is present in the cell-free protein synthesis system at a concentration of from about 0 to 50 g / L, wherein the ammonium source (if present) comprises ammonium sulfate, wherein the ammonium source (if present) is present in the cell-free protein synthesis system at a concentration of from 2 to 20 mM, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, wherein the potassium glutamate (if present) is present in the cell-free protein synthesis system at a concentration of from about 10 to 270 mM, wherein the potassium gluconate (if present) is present in the cell-free protein synthesis system at a concentration of from about 10 to 250 mM, wherein the magnesium source (if present) comprises magnesium glutamate, wherein the magnesium source is present in the cell-free protein synthesis system at a concentration of from 0 to about 10 mM, wherein the phosphate source (if present) comprises potassium phosphate, wherein the phosphate source (if present) is present in the cell-free protein synthesis system at a concentration of from about 5 to about 23.5 mM, wherein the sulfur source (if present) comprises ammonium sulfate, wherein the sulfur source (if present) is present in the cell-free protein synthesis system at a concentration of from 2 to 20 mM, wherein the buffer (if present) comprises 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, and wherein the buffer is present in the cell-free protein synthesis system at a concentration of from 50 to 300 mM.

[0060] In some embodiments, the concentration of the cell-free cell extract in the cell-free protein synthesis system is from 10% to 70% (v / v). In some embodiments, the concentration of the cell-free cell extract in the cell-free protein synthesis system is from 12% to 66% (v / v). In some embodiments, the concentration of the cell-free cell extract in the cell-free protein synthesis system is from 14% to 62% (v / v). In some embodiments, the concentration of the cell-free cell extract in the cell-free protein synthesis system is from 16% to 58% (v / v). In some embodiments, the concentration of the cell-free cell extract in the cell-free protein synthesis system is from 18% to 54% (v / v). In some embodiments, the concentration of the cell-free cell extract in the cell-free protein synthesis system is from 20% to 50% (v / v). In some embodiments, the concentration of the cell-free cell extract in the cell-free protein synthesis system is from 22% to 46% (v / v). In some embodiments, the concentration of the cell-free cell extract in the cell-free protein synthesis system is from 24% to 42% (v / v). In some embodiments, the concentration of the cell-free cell extract in the cell-free protein synthesis system is from 26% to 38% (v / v). In some embodiments, the concentration of the cell-free cell extract in the cell-free protein synthesis system is from 28% to 34% (v / v). In some embodiments, the concentration of the cell-free cell extract in the cell-free protein synthesis system is from 29% to 32% (v / v). In some embodiments, the concentration of the cell-free cell extract in the cell-free protein synthesis system is from 29% to 31% (v / v). In some embodiments, the concentration of the cell-free cell extract in the cell-free protein synthesis system is 30% (v / v). In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, and wherein the concentration of the cell-free cell extract in the cell-free protein synthesis system is from 10% to 70% (v / v).

[0061] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the cell-free protein synthesis system is maintained at a temperature below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; (g) a sulfur source; and (h) a buffer, wherein the molecular crowder is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowder (if present) in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the concentration of the carbon source (if present) in the cell-free protein synthesis system is from about 0 to 50 g / L, wherein the ammonium source (if present) comprises ammonium sulfate, wherein the concentration of the ammonium source (if present) in the cell-free protein synthesis system is from 2 to 20 mM, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, wherein the concentration of potassium glutamate (if present) in the cell-free protein synthesis system is from about 10 to 270 mM, wherein the concentration of potassium gluconate (if present) in the cell-free protein synthesis system is from about 10 to 250 mM, wherein the magnesium source (if present) comprises magnesium glutamate, wherein the concentration of the magnesium source (if present) in the cell-free protein synthesis system is from 0 to about 10 mM, wherein the phosphate source (if present) comprises potassium phosphate, wherein the concentration of the phosphate source in the cell-free protein synthesis system is from about 5 to about 23.5 mM, wherein the sulfur source (if present) comprises ammonium sulfate, wherein the concentration of the sulfur source (if present) in the cell-free protein synthesis system is from 2 to 20 mM, wherein the buffer (if present) comprises 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, wherein the concentration of the buffer in the cell-free protein synthesis system is from 50 to 300 mM, and wherein the concentration of the cell-free cell extract in the cell-free protein synthesis system is from 10% to 70% (v / v).

[0062] In some embodiments, the concentration of the DNA or RNA encoding the target protein in the cell-free protein synthesis system is from 1 to 100 μg / mL. In some embodiments, the concentration of the DNA or RNA encoding the target protein in the cell-free protein synthesis system is from 2 to 90 μg / mL. In some embodiments, the concentration of the DNA or RNA encoding the target protein in the cell-free protein synthesis system is from 3 to 80 μg / mL. In some embodiments, the concentration of the DNA or RNA encoding the target protein in the cell-free protein synthesis system is from 4 to 70 μg / mL. In some embodiments, the concentration of the DNA or RNA encoding the target protein in the cell-free protein synthesis system is from 5 to 60 μg / mL. In some embodiments, the concentration of the DNA or RNA encoding the target protein in the cell-free protein synthesis system is from 6 to 50 μg / mL. In some embodiments, the concentration of the DNA or RNA encoding the target protein in the cell-free protein synthesis system is from 7 to 40 μg / mL. In some embodiments, the concentration of the DNA or RNA encoding the target protein in the cell-free protein synthesis system is from 8 to 30 μg / mL. In some embodiments, the concentration of the DNA or RNA encoding the target protein in the cell-free protein synthesis system is from 9 to 20 μg / mL. In some embodiments, the concentration of the DNA or RNA encoding the target protein in the cell-free protein synthesis system is from 9 to 15 μg / mL. In some embodiments, the concentration of the DNA or RNA encoding the target protein in the cell-free protein synthesis system is from 9.5 to 12.5 μg / mL. In some embodiments, the concentration of the DNA or RNA encoding the target protein in the cell-free protein synthesis system is from 9.5 to 11.5 μg / mL. In some embodiments, the concentration of the DNA or RNA encoding the target protein in the cell-free protein synthesis system is from 9.5 to 11 μg / mL. In some embodiments, the concentration of the DNA or RNA encoding the target protein in the cell-free protein synthesis system is 10 μg / mL. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a target protein, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, and wherein the concentration of the DNA or RNA encoding the target protein in the cell-free protein synthesis system is from 1 to 100 μg / mL.

[0063] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the cell-free protein synthesis system is maintained at a temperature below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowder (if present) in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the concentration of the carbon source in the cell-free protein synthesis system is from about 0 to 50 g / L, wherein the ammonium source (if present) comprises ammonium sulfate, wherein the concentration of the ammonium source in the cell-free protein synthesis system is from 2 to 20 mM, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, wherein the concentration of potassium glutamate (if present) in the cell-free protein synthesis system is from about 10 to 270 mM, wherein the concentration of potassium gluconate in the cell-free protein synthesis system is from about 10 to 250 mM, wherein the magnesium source (if present) comprises magnesium glutamate, wherein the concentration of the magnesium source (if present) in the cell-free protein synthesis system is from 0 to about 10 mM, wherein the phosphate source (if present) comprises potassium phosphate, wherein the concentration of the phosphate source (if present) in the cell-free protein synthesis system is from about 5 to about 23.5 mM, wherein the sulfur source (if present) comprises ammonium sulfate, wherein the concentration of the sulfur source in the cell-free protein synthesis system is from 2 to 20 mM, wherein the buffer (if present) comprises 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, wherein the concentration of the buffer in the cell-free protein synthesis system is from 50 to 300 mM, wherein the concentration of the cell-free cell extract in the cell-free protein synthesis system is from 10% to 70% (v / v), and wherein the DNA or RNA encoding the protein of interest has a concentration in the cell-free protein synthesis system of from 1 to 100 μg / mL.

[0064] In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to the lysed bacteria, wherein the concentration of the free amino acids in the cell-free protein synthesis system is less than 2 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to the lysed bacteria, wherein the concentration of the free amino acids in the cell-free protein synthesis system is less than 1.9 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to the lysed bacteria, wherein the concentration of the free amino acids in the cell-free protein synthesis system is less than 1.8 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to the lysed bacteria, wherein the concentration of the free amino acids in the cell-free protein synthesis system is less than 1.7 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to the lysed bacteria, wherein the concentration of the free amino acids in the cell-free protein synthesis system is less than 1.6 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to the lysed bacteria, wherein the concentration of the free amino acids in the cell-free protein synthesis system is less than 1.5 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to the lysed bacteria, wherein the concentration of the free amino acids in the cell-free protein synthesis system is less than 1.4 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to the lysed bacteria, wherein the concentration of the free amino acids in the cell-free protein synthesis system is less than 1.3 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to the lysed bacteria, wherein the concentration of the free amino acids in the cell-free protein synthesis system is less than 1.2 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to the lysed bacteria, wherein the concentration of the free amino acids in the cell-free protein synthesis system is less than 1.1 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to the lysed bacteria, wherein the concentration of the free amino acids in the cell-free protein synthesis system is less than 1 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to the lysed bacteria, wherein the concentration of the free amino acids in the cell-free protein synthesis system is less than 0.9 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to the lysed bacteria, wherein the concentration of the free amino acids in the cell-free protein synthesis system is less than 0.8 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to the lysed bacteria, wherein the concentration of the free amino acids in the cell-free protein synthesis system is less than 0.7 mM.In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to the lysed bacteria, wherein the concentration of the free amino acids in the cell-free protein synthesis system is less than 0.6 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to the lysed bacteria, wherein the concentration of the free amino acids in the cell-free protein synthesis system is less than 0.5 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to the lysed bacteria, wherein the concentration of the free amino acids in the cell-free protein synthesis system is less than 0.4 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to the lysed bacteria, wherein the concentration of the free amino acids in the cell-free protein synthesis system is less than 0.3 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to the lysed bacteria, wherein the concentration of the free amino acids in the cell-free protein synthesis system is less than 0.2 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to the lysed bacteria, wherein the concentration of the free amino acids in the cell-free protein synthesis system is less than 0.1 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to the lysed bacteria, wherein the concentration of the free amino acids in the cell-free protein synthesis system is less than 0.05 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to the lysed bacteria, wherein the concentration of the free amino acids in the cell-free protein synthesis system is less than 0.025 mM. In some embodiments, the cell-free protein synthesis system comprises free amino acids endogenous to the lysed bacteria, wherein the concentration of the free amino acids in the cell-free protein synthesis system is less than 0.01 mM. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless the free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the cell-free protein synthesis system comprises free amino acids endogenous to the lysed bacteria, and wherein the concentration of the free amino acids in the cell-free protein synthesis system is less than 2 mM.

[0065] In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the cell-free protein synthesis system is maintained at a temperature below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowder (if present) in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the concentration of the carbon source (if present) in the cell-free protein synthesis system is from about 0 to 50 g / L, wherein the ammonium source (if present) comprises ammonium sulfate, wherein the concentration of the ammonium source (if present) in the cell-free protein synthesis system is from 2 to 20 mM, wherein the potassium source is selected from potassium glutamate and potassium gluconate, wherein the concentration of potassium glutamate (if present) in the cell-free protein synthesis system is from about 10 to 270 mM, wherein the concentration of potassium gluconate in the cell-free protein synthesis system is from about 10 to 250 mM, wherein the magnesium source (if present) comprises magnesium glutamate, wherein the concentration of the magnesium source in the cell-free protein synthesis system is from 0 to about 10 mM, wherein the phosphate source (if present) comprises potassium phosphate, wherein the concentration of the phosphate source (if present) in the cell-free protein synthesis system is from about 5 to about 23.5 mM, wherein the sulfur source (if present) comprises ammonium sulfate, wherein the concentration of the sulfur source (if present) in the cell-free protein synthesis system is from 2 to 20 mM, wherein the buffer (if present) comprises 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, wherein the concentration of the buffer in the cell-free protein synthesis system is from 50 to 300 mM, wherein the cell-free cell extract has a concentration in the cell-free protein synthesis system of from 10% to 70% (v / v), wherein the DNA or RNA encoding the protein of interest has a concentration in the cell-free protein synthesis system of from 1 to 100 μg / mL, wherein the cell-free protein synthesis system contains free amino acids endogenous to the lysed bacteria, and wherein the concentration of free amino acids in the cell-free protein synthesis system is less than 2 mM.

[0066] In some embodiments, the cell-free protein synthesis system comprises free nucleotides endogenous to the lysed bacteria, wherein the concentration of the free nucleotides in the cell-free protein synthesis system is less than 1.5 mM. In some embodiments, the cell-free protein synthesis system comprises free nucleotides endogenous to the lysed bacteria, wherein the concentration of the free nucleotides in the cell-free protein synthesis system is less than 1.4 mM. In some embodiments, the cell-free protein synthesis system comprises free nucleotides endogenous to the lysed bacteria, wherein the concentration of the free nucleotides in the cell-free protein synthesis system is less than 1.3 mM. In some embodiments, the cell-free protein synthesis system comprises free nucleotides endogenous to the lysed bacteria, wherein the concentration of the free nucleotides in the cell-free protein synthesis system is less than 1.2 mM. In some embodiments, the cell-free protein synthesis system comprises free nucleotides endogenous to the lysed bacteria, wherein the concentration of the free nucleotides in the cell-free protein synthesis system is less than 1.1 mM. In some embodiments, the cell-free protein synthesis system comprises free nucleotides endogenous to the lysed bacteria, wherein the concentration of the free nucleotides in the cell-free protein synthesis system is less than 1 mM. In some embodiments, the cell-free protein synthesis system comprises free nucleotides endogenous to the lysed bacteria, wherein the concentration of the free nucleotides in the cell-free protein synthesis system is less than 0.9 mM. In some embodiments, the cell-free protein synthesis system comprises free nucleotides endogenous to the lysed bacteria, wherein the concentration of the free nucleotides in the cell-free protein synthesis system is less than 0.8 mM. In some embodiments, the cell-free protein synthesis system comprises free nucleotides endogenous to the lysed bacteria, wherein the concentration of the free nucleotides in the cell-free protein synthesis system is less than 0.7 mM. In some embodiments, the cell-free protein synthesis system comprises free nucleotides endogenous to the lysed bacteria, wherein the concentration of the free nucleotides in the cell-free protein synthesis system is less than 0.6 mM. In some embodiments, the cell-free protein synthesis system comprises free nucleotides endogenous to the lysed bacteria, wherein the concentration of the free nucleotides in the cell-free protein synthesis system is less than 0.5 mM. In some embodiments, the cell-free protein synthesis system comprises free nucleotides endogenous to the lysed bacteria, wherein the concentration of the free nucleotides in the cell-free protein synthesis system is less than 0.25 mM. In some embodiments, the cell-free protein synthesis system comprises free nucleotides endogenous to the lysed bacteria, wherein the concentration of the free nucleotides in the cell-free protein synthesis system is less than 0.125 mM. In some embodiments, the cell-free protein synthesis system comprises free nucleotides endogenous to the lysed bacteria, wherein the concentration of the free nucleotides in the cell-free protein synthesis system is less than 0.1 mM.In some embodiments, the cell-free protein synthesis system comprises free nucleotides endogenous to the lysed bacteria, wherein the concentration of the free nucleotides in the cell-free protein synthesis system is less than 0.075 mM. In some embodiments, the cell-free protein synthesis system comprises free nucleotides endogenous to the lysed bacteria, wherein the concentration of the free nucleotides in the cell-free protein synthesis system is less than 0.05 mM. In some embodiments, the cell-free protein synthesis system comprises free nucleotides endogenous to the lysed bacteria, wherein the concentration of the free nucleotides in the cell-free protein synthesis system is less than 0.025 mM. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless the free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the cell-free protein synthesis system comprises free nucleotides endogenous to the lysed bacteria, and wherein the concentration of the free nucleotides in the cell-free protein synthesis system is less than 1.5 mM.

[0067] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the cell-free protein synthesis system is maintained at a temperature below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowder (if present) in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the concentration of the carbon source (if present) in the cell-free protein synthesis system is from about 0 to 50 g / L, wherein the ammonium source (if present) comprises ammonium sulfate, wherein the concentration of the ammonium source in the cell-free protein synthesis system is from 2 to 20 mM, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, wherein the concentration of potassium glutamate (if present) in the cell-free protein synthesis system is from about 10 to 270 mM, wherein the concentration of potassium gluconate (if present) in the cell-free protein synthesis system is from about 10 to 250 mM, wherein the magnesium source (if present) comprises magnesium glutamate, wherein the concentration of the magnesium source (if present) in the cell-free protein synthesis system is from 0 to about 10 mM, wherein the phosphate source (if present) comprises potassium phosphate, wherein the concentration of the phosphate source in the cell-free protein synthesis system is from about 5 to about 23.5 mM, wherein the sulfur source (if present) comprises ammonium sulfate, wherein the concentration of the sulfur source in the cell-free protein synthesis system is from 2 to 20 mM, wherein the buffer (if present) comprises 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, wherein the concentration of the buffer in the cell-free protein synthesis system is from 50 to 300 mM, wherein the concentration of the cell-free cell extract in the cell-free protein synthesis system is from 10% to 70% (v / v), wherein the concentration of the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is from 1 to 100 μg / mL, wherein the cell-free protein synthesis system comprises free amino acids endogenous to the lysed bacteria, wherein the concentration of the free amino acids in the cell-free protein synthesis system is less than 2 mM, wherein the cell-free protein synthesis system comprises free nucleotides endogenous to the lysed bacteria, and wherein the concentration of the free nucleotides in the cell-free protein synthesis system is less than 1.5 mM.

[0068] In some embodiments, the cell-free protein synthesis system comprises free NADH / NADPH endogenous to the lysed bacteria, wherein the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM. In some embodiments, the cell-free protein synthesis system comprises free NADH / NADPH endogenous to the lysed bacteria, wherein the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.35 mM. In some embodiments, the cell-free protein synthesis system comprises free NADH / NADPH endogenous to the lysed bacteria, wherein the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.3 mM. In some embodiments, the cell-free protein synthesis system comprises free NADH / NADPH endogenous to the lysed bacteria, wherein the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.25 mM. In some embodiments, the cell-free protein synthesis system comprises free NADH / NADPH endogenous to the lysed bacteria, wherein the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.2 mM. In some embodiments, the cell-free protein synthesis system comprises free NADH / NADPH endogenous to the lysed bacteria, wherein the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.15 mM. In some embodiments, the cell-free protein synthesis system comprises free NADH / NADPH endogenous to the lysed bacteria, wherein the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.1 mM. In some embodiments, the cell-free protein synthesis system comprises free NADH / NADPH endogenous to the lysed bacteria, wherein the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.08 mM. In some embodiments, the cell-free protein synthesis system comprises free NADH / NADPH endogenous to the lysed bacteria, wherein the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.06 mM. In some embodiments, the cell-free protein synthesis system comprises free NADH / NADPH endogenous to the lysed bacteria, wherein the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.04 mM. In some embodiments, the cell-free protein synthesis system comprises free NADH / NADPH endogenous to the lysed bacteria, wherein the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.02 mM.In some embodiments, the cell-free protein synthesis system comprises free NADH / NADPH endogenous to the lysed bacteria, wherein the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.01 mM. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless the free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, and wherein the cell-free protein synthesis system comprises free NADH / NADPH endogenous to the lysed bacteria, and wherein the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM.

[0069] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the cell-free protein synthesis system is maintained at a temperature below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder (if present) is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowder in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the concentration of the carbon source in the cell-free protein synthesis system is from about 0 to 50 g / L, wherein the ammonium source (if present) comprises ammonium sulfate, wherein the concentration of the ammonium source in the cell-free protein synthesis system is from 2 to 20 mM, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, wherein the concentration of potassium glutamate in the cell-free protein synthesis system is from about 10 to 270 mM, wherein the concentration of potassium gluconate in the cell-free protein synthesis system is from about 10 to 250 mM, wherein the magnesium source (if present) comprises magnesium glutamate, wherein the concentration of the magnesium source in the cell-free protein synthesis system is from 0 to about 10 mM, wherein the phosphate source comprises potassium phosphate, wherein the concentration of the phosphate source in the cell-free protein synthesis system is from about 5 to about 23.5 mM, wherein the sulfur source (if present) comprises ammonium sulfate, wherein the concentration of the sulfur source in the cell-free protein synthesis system is from 2 to 20 mM, wherein the buffer (if present) comprises 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, wherein the concentration of the buffer in the cell-free protein synthesis system is from 50 to 300 mM, wherein the cell-free cell extract has a concentration in the cell-free protein synthesis system of from 10% to 70% (v / v), wherein the DNA or RNA encoding the protein of interest has a concentration in the cell-free protein synthesis system of from 1 to 100 μg / mL, wherein the cell-free protein synthesis system comprises free amino acids endogenous to the lysed bacteria, wherein the free amino acids have a concentration in the cell-free protein synthesis system of less than 2 mM, wherein the cell-free protein synthesis system comprises free nucleotides endogenous to the lysed bacteria, wherein the free nucleotides have a concentration in the cell-free protein synthesis system of less than 1.5 mM, wherein the cell-free protein synthesis system comprises free NADH / NADPH endogenous to the lysed bacteria, and wherein the free NADH / NADPH has a concentration in the cell-free protein synthesis system of less than 0.4 mM.

[0070] In some embodiments, the cell-free protein synthesis system does not contain tryptone. In some embodiments, the cell-free protein synthesis system does not contain yeast extract. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless the free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, and wherein the cell-free protein synthesis system does not contain tryptone. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless the free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, and wherein the cell-free protein synthesis system does not contain yeast extract.

[0071] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the cell-free protein synthesis system is maintained at a temperature below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowder (if present) in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the concentration of the carbon source in the cell-free protein synthesis system is from about 0 to 50 g / L, wherein the ammonium source (if present) comprises ammonium sulfate, wherein the concentration of the ammonium source (if present) in the cell-free protein synthesis system is from 2 to 20 mM, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, wherein the concentration of potassium glutamate in the cell-free protein synthesis system is from about 10 to 270 mM, wherein the concentration of potassium gluconate (if present) in the cell-free protein synthesis system is from about 10 to 250 mM, wherein the magnesium source comprises magnesium glutamate, wherein the concentration of the magnesium source in the cell-free protein synthesis system is from 0 to about 10 mM, wherein the phosphate source (if present) comprises potassium phosphate, wherein the concentration of the phosphate source in the cell-free protein synthesis system is from about 5 to about 23.5 mM, wherein the sulfur source (if present) comprises ammonium sulfate, wherein the concentration of the sulfur source in the cell-free protein synthesis system is from 2 to 20 mM, wherein the buffer (if present) comprises 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, wherein the concentration of the buffer in the cell-free protein synthesis system is from 50 to 300 mM, wherein the cell-free cell extract has a concentration in the cell-free protein synthesis system of from 10% to 70% (v / v), wherein the DNA or RNA encoding the protein of interest has a concentration in the cell-free protein synthesis system of from 1 to 100 μg / mL, wherein the cell-free protein synthesis system contains free amino acids endogenous to the lysed bacteria, wherein the free amino acids have a concentration in the cell-free protein synthesis system of less than 2 mM, wherein the cell-free protein synthesis system contains free nucleotides endogenous to the lysed bacteria, wherein the free nucleotides have a concentration in the cell-free protein synthesis system of less than 1.5 mM, wherein the cell-free protein synthesis system contains free NADH / NADPH endogenous to the lysed bacteria, wherein the free NADH / NADPH has a concentration in the cell-free protein synthesis system of less than 0.4 mM, wherein the cell-free protein synthesis system does not contain tryptone, and wherein the cell-free protein synthesis system does not contain yeast extract. Minimal medium

[0072] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria. In some embodiments, the minimal medium comprises at least one of the following: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate. In some embodiments, the minimal medium comprises at least two of the following: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate. In some embodiments, the minimal medium comprises at least three of the following: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate. In some embodiments, the minimal medium comprises at least four of the following: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate. In some embodiments, the minimal medium comprises at least five of the following: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate. In some embodiments, the minimal medium comprises at least six of the following: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate. In some embodiments, the minimal medium comprises all of the following: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate.In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, and wherein the minimal medium comprises at least one of the following: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate.

[0073] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the cell-free protein synthesis system is maintained at a temperature below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowder (if present) in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the concentration of the carbon source in the cell-free protein synthesis system is from about 0 to 50 g / L, wherein the ammonium source (if present) comprises ammonium sulfate, wherein the concentration of the ammonium source in the cell-free protein synthesis system is from 2 to 20 mM, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, wherein the concentration of potassium glutamate in the cell-free protein synthesis system is from about 10 to 270 mM, wherein the concentration of potassium gluconate in the cell-free protein synthesis system is from about 10 to 250 mM, wherein the magnesium source (if present) comprises magnesium glutamate, wherein the concentration of the magnesium source in the cell-free protein synthesis system is from 0 to about 10 mM, wherein the phosphate source (if present) comprises potassium phosphate, wherein the concentration of the phosphate source in the cell-free protein synthesis system is from about 5 to about 23.5 mM, wherein the sulfur source (if present) comprises ammonium sulfate, wherein the concentration of the sulfur source in the cell-free protein synthesis system is from 2 to 20 mM, wherein the buffer (if present) comprises 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, wherein the concentration of the buffer in the cell-free protein synthesis system is from 50 to 300 mM, wherein the cell-free cell extract has a concentration in the cell-free protein synthesis system of from 10% to 70% (v / v), wherein the DNA or RNA encoding the protein of interest has a concentration in the cell-free protein synthesis system of from 1 to 100 μg / mL, wherein the cell-free protein synthesis system contains free amino acids endogenous to the lysed bacteria, wherein the free amino acids have a concentration in the cell-free protein synthesis system of less than 2 mM, wherein the cell-free protein synthesis system contains free nucleotides endogenous to the lysed bacteria, wherein the free nucleotides have a concentration in the cell-free protein synthesis system of less than 1.5 mM, wherein the cell-free protein synthesis system contains free NADH / NADPH endogenous to the lysed bacteria, wherein the free NADH / NADPH has a concentration in the cell-free protein synthesis system of less than 0.4 mM, wherein the cell-free protein synthesis system does not contain tryptone, wherein the cell-free protein synthesis system does not contain yeast extract, and wherein the basal medium contains at least one of the following: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate.

[0074] In some embodiments, the carbon source is selected from D-glucose and glycerol. In some embodiments, the concentration of D-glucose in the basal medium is from 0.1% to 2.5% (w / v). In some embodiments, the concentration of D-glucose in the basal medium is from 0.2% to 2% (w / v). In some embodiments, the concentration of D-glucose in the basal medium is from 0.3% to 1.5% (w / v). In some embodiments, the concentration of D-glucose in the basal medium is from 0.4% to 1% (w / v). In some embodiments, the concentration of D-glucose in the basal medium is from 0.4% to 0.8% (w / v). In some embodiments, the concentration of D-glucose in the basal medium is from 0.4% to 0.6% (w / v). In some embodiments, the concentration of D-glucose in the basal medium is 0.5% (w / v). In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a basal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, and wherein the basal medium comprises at least one of the following: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the carbon source is selected from D-glucose and glycerol, and wherein the concentration of D-glucose in the basal medium is from 0.1% to 2.5% (w / v).

[0075] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the cell-free protein synthesis system is maintained at a temperature below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder (if present) is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowder (if present) in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the concentration of the carbon source in the cell-free protein synthesis system is from about 0 to 50 g / L, wherein the ammonium source (if present) comprises ammonium sulfate, wherein the concentration of the ammonium source (if present) in the cell-free protein synthesis system is from 2 to 20 mM, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, wherein the concentration of potassium glutamate in the cell-free protein synthesis system is from about 10 to 27 mM, wherein the concentration of potassium gluconate in the cell-free protein synthesis system is from about 10 to 25 mM, wherein the magnesium source (if present) comprises magnesium glutamate, wherein the concentration of the magnesium source in the cell-free protein synthesis system is from 0 to about 10 mM, wherein the phosphate source (if present) comprises potassium phosphate, wherein the concentration of the phosphate source in the cell-free protein synthesis system is from about 5 to about 23.5 mM, wherein the sulfur source comprises ammonium sulfate, wherein the concentration of the sulfur source in the cell-free protein synthesis system is from 2 to 20 mM, wherein the buffer (if present) comprises 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, wherein the concentration of the buffer in the cell-free protein synthesis system is from 50 to 300 mM, wherein the cell-free cell extract has a concentration in the cell-free protein synthesis system of from 10% to 70% (v / v), wherein the DNA or RNA encoding the protein of interest has a concentration in the cell-free protein synthesis system of from 1 to 100 μg / mL, wherein the cell-free protein synthesis system contains free amino acids endogenous to the lysed bacteria, wherein the free amino acids have a concentration in the cell-free protein synthesis system of less than 2 mM, wherein the cell-free protein synthesis system contains free nucleotides endogenous to the lysed bacteria, wherein the free nucleotides have a concentration in the cell-free protein synthesis system of less than 1.5 mM, wherein the cell-free protein synthesis system contains free NADH / NADPH endogenous to the lysed bacteria, wherein the free NADH / NADPH has a concentration in the cell-free protein synthesis system of less than 0.4 mM, wherein the cell-free protein synthesis system does not contain tryptone, wherein the cell-free protein synthesis system does not contain yeast extract, wherein the basal medium comprises at least one of the following: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the carbon source is selected from D-glucose and glycerol, and wherein the D-glucose has a concentration in the basal medium of from 0.1% to 2.5% (w / v).

[0076] In some embodiments, the carbon source is selected from D-glucose and glycerol. In some embodiments, the concentration of the glycerol in the basal medium is from 0.25% to 25% (w / v). In some embodiments, the concentration of the glycerol in the basal medium is from 0.5% to 22.5% (w / v). In some embodiments, the concentration of the glycerol in the basal medium is from 0.75% to 20% (w / v). In some embodiments, the concentration of the glycerol in the basal medium is from 1% to 17.5% (w / v). In some embodiments, the concentration of the glycerol in the basal medium is from 1.25% to 15% (w / v). In some embodiments, the concentration of the glycerol in the basal medium is from 1.5% to 12.5% (w / v). In some embodiments, the concentration of the glycerol in the basal medium is from 1.75% to 10% (w / v). In some embodiments, the concentration of the glycerol in the basal medium is from 2% to 7.5% (w / v). In some embodiments, the concentration of the glycerol in the basal medium is from 2.25% to 5% (w / v). In some embodiments, the concentration of the glycerol in the basal medium is from 2.25% to 3.75% (w / v). In some embodiments, the concentration of the glycerol in the basal medium is from 2.25% to 2.75% (w / v). In some embodiments, the concentration of the glycerol in the basal medium is 2.5% (w / v). In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a basal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless such free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the basal medium comprises at least one of the following: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the carbon source is selected from D-glucose and glycerol, and wherein the concentration of the glycerol in the basal medium is from 0.25% to 25% (w / v).

[0077] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the cell-free protein synthesis system is maintained at a temperature below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder (if present) is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowder (if present) in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the concentration of the carbon source in the cell-free protein synthesis system is from about 0 to 50 g / L, wherein the ammonium source (if present) comprises ammonium sulfate, wherein the concentration of the ammonium source (if present) in the cell-free protein synthesis system is from 2 to 20 mM, wherein the potassium source is selected from potassium glutamate and potassium gluconate, wherein the concentration of potassium glutamate (if present) in the cell-free protein synthesis system is from about 10 to 270 mM, wherein the concentration of potassium gluconate in the cell-free protein synthesis system is from about 10 to 250 mM, wherein the magnesium source (if present) comprises magnesium glutamate, wherein the concentration of the magnesium source in the cell-free protein synthesis system is from 0 to about 10 mM, wherein the phosphate source (if present) comprises potassium phosphate, wherein the concentration of the phosphate source in the cell-free protein synthesis system is from about 5 to about 23.5 mM, wherein the sulfur source (if present) comprises ammonium sulfate, wherein the concentration of the sulfur source in the cell-free protein synthesis system is from 2 to 20 mM, wherein the buffer (if present) comprises 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, wherein the concentration of the buffer in the cell-free protein synthesis system is from 50 to 300 mM, wherein the concentration of the cell-free cell extract in the cell-free protein synthesis system is from 10% to 70% (v / v), wherein the concentration of the DNA or RNA encoding the target protein in the cell-free protein synthesis system is from 1 to 100 μg / mL, wherein the cell-free protein synthesis system contains free amino acids endogenous to the lysed bacteria, wherein the concentration of the free amino acids in the cell-free protein synthesis system is less than 2 mM, wherein the cell-free protein synthesis system contains free nucleotides endogenous to the lysed bacteria, wherein the concentration of the free nucleotides in the cell-free protein synthesis system is less than 1.5 mM, wherein the cell-free protein synthesis system contains free NADH / NADPH endogenous to the lysed bacteria, wherein the concentration of the free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM, wherein the cell-free protein synthesis system does not contain tryptone, wherein the cell-free protein synthesis system does not contain yeast extract, wherein the basal medium comprises at least one of the following: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the carbon source is selected from D-glucose and glycerol, wherein the concentration of the D-glucose in the basal medium is from 0.1% to 2.5% (w / v), and wherein the concentration of the glycerol in the basal medium is from 0.25% to 25% (w / v).

[0078] In some embodiments, the concentration of sodium succinate in the basal medium is from 0.05% to 5% (w / v). In some embodiments, the concentration of sodium succinate in the basal medium is from 0.1% to 4% (w / v). In some embodiments, the concentration of sodium succinate in the basal medium is from 0.15% to 3% (w / v). In some embodiments, the concentration of sodium succinate in the basal medium is from 0.2% to 2% (w / v). In some embodiments, the concentration of sodium succinate in the basal medium is from 0.25% to 1% (w / v). In some embodiments, the concentration of sodium succinate in the basal medium is from 0.25% to 0.75% (w / v). In some embodiments, the concentration of sodium succinate in the basal medium is from 0.25% to 0.5% (w / v). In some embodiments, the concentration of sodium succinate in the basal medium is 0.375% (w / v). In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a basal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless the free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the basal medium comprises at least one of the following: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, and wherein the concentration of sodium succinate in the basal medium is from 0.05% to 5% (w / v).

[0079] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the cell-free protein synthesis system is maintained at a temperature below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowding agent; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowding agent is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowding agent (if present) in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the concentration of the carbon source in the cell-free protein synthesis system is from about 0 to 50 g / L, wherein the ammonium source (if present) comprises ammonium sulfate, wherein the concentration of the ammonium source in the cell-free protein synthesis system is from 2 to 20 mM, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, wherein the concentration of potassium glutamate (if present) in the cell-free protein synthesis system is from about 10 to 270 mM, wherein the concentration of potassium gluconate (if present) in the cell-free protein synthesis system is from about 10 to 250 mM, wherein the magnesium source (if present) comprises magnesium glutamate, wherein the concentration of the magnesium source in the cell-free protein synthesis system is from 0 to about 10 mM, wherein the phosphate source (if present) comprises potassium phosphate, wherein the concentration of the phosphate source in the cell-free protein synthesis system is from about 5 to about 23.5 mM, wherein the sulfur source (if present) comprises ammonium sulfate, wherein the concentration of the sulfur source in the cell-free protein synthesis system is from 2 to 20 mM, wherein the buffer (if present) comprises 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, wherein the concentration of the buffer in the cell-free protein synthesis system is from 50 to 300 mM, wherein the cell-free cell extract has a concentration in the cell-free protein synthesis system of from 10% to 70% (v / v), wherein the DNA or RNA encoding the protein of interest has a concentration in the cell-free protein synthesis system of from 1 to 100 μg / mL, wherein the cell-free protein synthesis system contains free amino acids endogenous to the lysed bacteria, wherein the free amino acids have a concentration in the cell-free protein synthesis system of less than 2 mM, wherein the cell-free protein synthesis system contains free nucleotides endogenous to the lysed bacteria, wherein the free nucleotides have a concentration in the cell-free protein synthesis system of less than 1.5 mM, wherein the cell-free protein synthesis system contains free NADH / NADPH endogenous to the lysed bacteria, wherein the free NADH / NADPH has a concentration in the cell-free protein synthesis system of less than 0.4 mM, wherein the cell-free protein synthesis system does not contain tryptone, wherein the cell-free protein synthesis system does not contain yeast extract, wherein the basal medium comprises at least one of the following: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the carbon source is selected from D-glucose and glycerol, wherein the D-glucose has a concentration in the basal medium of from 0.1% to 2.5% (w / v), wherein the glycerol has a concentration in the basal medium of from 0.25% to 25% (w / v), and wherein the sodium succinate has a concentration in the basal medium of from 0.05% to 5% (w / v).

[0080] In some embodiments, the phosphate source includes sodium phosphate buffer or potassium phosphate buffer. In some embodiments, the concentration of the potassium phosphate buffer in the basal medium is from 5 to 250 mM. In some embodiments, the concentration of the potassium phosphate buffer in the basal medium is from 10 to 200 mM. In some embodiments, the concentration of the potassium phosphate buffer in the basal medium is from 15 to 150 mM. In some embodiments, the concentration of the potassium phosphate buffer in the basal medium is from 20 to 100 mM. In some embodiments, the concentration of the potassium phosphate buffer in the basal medium is from 25 to 75 mM. In some embodiments, the concentration of the potassium phosphate buffer in the basal medium is from 37.5 to 62.5 mM. In some embodiments, the concentration of the potassium phosphate buffer in the basal medium is from 45 to 55 mM. In some embodiments, the concentration of the potassium phosphate buffer in the basal medium is from 47.5 to 52.5 mM. In some embodiments, the concentration of the potassium phosphate buffer in the basal medium is 50 mM. In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a basal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the basal medium comprises at least one of the following: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the phosphate source includes sodium phosphate buffer or potassium phosphate buffer, and wherein the concentration of the potassium phosphate buffer in the basal medium is from 5 to 250 mM.

[0081] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the cell-free protein synthesis system is maintained at a temperature below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowder (if present) in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the concentration of the carbon source in the cell-free protein synthesis system is from about 0 to 50 g / L, wherein the ammonium source (if present) comprises ammonium sulfate, wherein the concentration of the ammonium source in the cell-free protein synthesis system is from 2 to 20 mM, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, wherein the concentration of potassium glutamate in the cell-free protein synthesis system is from about 10 to 270 mM, wherein the concentration of potassium gluconate in the cell-free protein synthesis system is from about 10 to 250 mM, wherein the magnesium source (if present) comprises magnesium glutamate, wherein the concentration of the magnesium source in the cell-free protein synthesis system is from 0 to about 10 mM, wherein the phosphate source (if present) comprises potassium phosphate, wherein the concentration of the phosphate source in the cell-free protein synthesis system is from about 5 to about 23.5 mM, wherein the sulfur source (if present) comprises ammonium sulfate, wherein the concentration of the sulfur source in the cell-free protein synthesis system is from 2 to 20 mM, wherein the buffer (if present) comprises 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, wherein the concentration of the buffer in the cell-free protein synthesis system is from 50 to 300 mM, wherein the cell-free cell extract in the cell-free protein synthesis system is at a concentration of from 10% to 70% (v / v), wherein the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is at a concentration of from 1 to 100 μg / mL, wherein the cell-free protein synthesis system contains free amino acids endogenous to the lysed bacteria, wherein the free amino acids in the cell-free protein synthesis system are at a concentration of less than 2 mM, wherein the cell-free protein synthesis system contains free nucleotides endogenous to the lysed bacteria, wherein the free nucleotides in the cell-free protein synthesis system are at a concentration of less than 1.5 mM, wherein the cell-free protein synthesis system contains free NADH / NADPH endogenous to the lysed bacteria, wherein the free NADH / NADPH in the cell-free protein synthesis system is at a concentration of less than 0.4 mM, wherein the cell-free protein synthesis system does not contain tryptone, wherein the cell-free protein synthesis system does not contain yeast extract, wherein the basal medium comprises at least one of the following: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the carbon source is selected from D-glucose and glycerol, wherein the D-glucose in the basal medium is at a concentration of from 0.1% to 2.5% (w / v), wherein the glycerol in the basal medium is at a concentration of from 0.25% to 25% (w / v), wherein the sodium succinate in the basal medium is at a concentration of from 0.05% to 5% (w / v), wherein the phosphate source comprises sodium phosphate buffer or potassium phosphate buffer, and wherein the potassium phosphate buffer in the basal medium is at a concentration of from 5 to 250 mM.

[0082] In some embodiments, the pH of the potassium phosphate buffer in the basal medium is from 6.7 to 7.7. In some embodiments, the pH of the potassium phosphate buffer in the basal medium is from 6.8 to 7.6. In some embodiments, the pH of the potassium phosphate buffer in the basal medium is from 6.9 to 7.5. In some embodiments, the pH of the potassium phosphate buffer in the basal medium is from 7 to 7.4. In some embodiments, the pH of the potassium phosphate buffer in the basal medium is from 7.1 to 7.3. In some embodiments, the pH of the potassium phosphate buffer in the basal medium is 7.2. In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a basal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless the free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the basal medium comprises at least one of the following: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the phosphate source comprises a sodium phosphate buffer or a potassium phosphate buffer, and wherein the pH of the potassium phosphate buffer in the basal medium is from 6.7 to 7.7.

[0083] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the cell-free protein synthesis system is maintained at a temperature below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowder (if present) in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the concentration of the carbon source in the cell-free protein synthesis system is from about 0 to 50 g / L, wherein the ammonium source (if present) comprises ammonium sulfate, wherein the concentration of the ammonium source in the cell-free protein synthesis system is from 2 to 20 mM, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, wherein the concentration of potassium glutamate in the cell-free protein synthesis system is from about 10 to 270 mM, wherein the concentration of potassium gluconate in the cell-free protein synthesis system is from about 10 to 250 mM, wherein the magnesium source (if present) comprises magnesium glutamate, wherein the concentration of the magnesium source in the cell-free protein synthesis system is from 0 to about 10 mM, wherein the phosphate source comprises potassium phosphate, wherein the concentration of the phosphate source in the cell-free protein synthesis system is from about 5 to about 23.5 mM, wherein the sulfur source (if present) comprises ammonium sulfate, wherein the concentration of the sulfur source in the cell-free protein synthesis system is from 2 to 20 mM, wherein the buffer (if present) comprises 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, wherein the concentration of the buffer in the cell-free protein synthesis system is from 50 to 300 mM, wherein the cell-free cell extract has a concentration in the cell-free protein synthesis system of from 10% to 70% (v / v), wherein the DNA or RNA encoding the protein of interest has a concentration in the cell-free protein synthesis system of from 1 to 100 μg / mL, wherein the cell-free protein synthesis system contains free amino acids endogenous to the lysed bacteria, wherein the free amino acids have a concentration in the cell-free protein synthesis system of less than 2 mM, wherein the cell-free protein synthesis system contains free nucleotides endogenous to the lysed bacteria, wherein the free nucleotides have a concentration in the cell-free protein synthesis system of less than 1.5 mM, wherein the cell-free protein synthesis system contains free NADH / NADPH endogenous to the lysed bacteria, wherein the free NADH / NADPH has a concentration in the cell-free protein synthesis system of less than 0.4 mM, wherein the cell-free protein synthesis system does not contain tryptone, wherein the cell-free protein synthesis system does not contain yeast extract, wherein the basal medium comprises at least one of the following: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the carbon source is selected from D-glucose and glycerol, wherein the D-glucose has a concentration in the basal medium of from 0.1% to 2.5% (w / v), wherein the glycerol has a concentration in the basal medium of from 0.25% to 25% (w / v), wherein the sodium succinate has a concentration in the basal medium of from 0.05% to 5% (w / v), wherein the phosphate source comprises sodium phosphate buffer or potassium phosphate buffer, wherein the potassium phosphate buffer has a concentration in the basal medium of from 5 to 250 mM, and wherein the pH of the potassium phosphate buffer in the basal medium is from 6.7 to 7.7.

[0084] In some embodiments, the ammonium source comprises ammonium chloride. In some embodiments, the concentration of ammonium chloride in the basal medium is from 5 to 250 mM. In some embodiments, the concentration of ammonium chloride in the basal medium is from 10 to 200 mM. In some embodiments, the concentration of ammonium chloride in the basal medium is from 15 to 150 mM. In some embodiments, the concentration of ammonium chloride in the basal medium is from 20 to 100 mM. In some embodiments, the concentration of ammonium chloride in the basal medium is from 25 to 75 mM. In some embodiments, the concentration of ammonium chloride in the basal medium is from 37.5 to 62.5 mM. In some embodiments, the concentration of ammonium chloride in the basal medium is from 45 to 55 mM. In some embodiments, the concentration of ammonium chloride in the basal medium is from 47.5 to 52.5 mM. In some embodiments, the concentration of ammonium chloride in the basal medium is 50 mM. In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a basal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the basal medium comprises at least one of the following: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the ammonium source comprises ammonium chloride, and wherein the concentration of ammonium chloride in the basal medium is from 5 to 250 mM.

[0085] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the cell-free protein synthesis system is maintained at a temperature below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowder (if present) in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the concentration of the carbon source in the cell-free protein synthesis system is from about 0 to 50 g / L, wherein the ammonium source (if present) comprises ammonium sulfate, wherein the concentration of the ammonium source (if present) in the cell-free protein synthesis system is from 2 to 20 mM, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, wherein the concentration of potassium glutamate in the cell-free protein synthesis system is from about 10 to 270 mM, wherein the concentration of potassium gluconate in the cell-free protein synthesis system is from about 10 to 250 mM, wherein the magnesium source (if present) comprises magnesium glutamate, wherein the concentration of the magnesium source in the cell-free protein synthesis system is from 0 to about 10 mM, wherein the phosphate source (if present) comprises potassium phosphate, wherein the concentration of the phosphate source in the cell-free protein synthesis system is from about 5 to about 23.5 mM, wherein the sulfur source comprises ammonium sulfate, wherein the concentration of the sulfur source in the cell-free protein synthesis system is from 2 to 20 mM, wherein the buffer comprises 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, wherein the concentration of the buffer in the cell-free protein synthesis system is from 50 to 300 mM, wherein the concentration of the cell-free cell extract in the cell-free protein synthesis system is from 10% to 70% (v / v), wherein the DNA or RNA encoding the target protein has a concentration in the cell-free protein synthesis system of from 1 to 100 μg / mL, wherein the cell-free protein synthesis system contains free amino acids endogenous to the lysed bacteria, wherein the free amino acids have a concentration in the cell-free protein synthesis system of less than 2 mM, wherein the cell-free protein synthesis system contains free nucleotides endogenous to the lysed bacteria, wherein the free nucleotides have a concentration in the cell-free protein synthesis system of less than 1.5 mM, wherein the cell-free protein synthesis system contains free NADH / NADPH endogenous to the lysed bacteria, wherein the free NADH / NADPH has a concentration in the cell-free protein synthesis system of less than 0.4 mM, wherein the cell-free protein synthesis system does not contain tryptone, wherein the cell-free protein synthesis system does not contain yeast extract, wherein the basal medium comprises at least one of the following: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the carbon source is selected from D-glucose and glycerol, wherein the D-glucose has a concentration in the basal medium of from 0.1% to 2.5% (w / v), wherein the glycerol has a concentration in the basal medium of from 0.25% to 25% (w / v), wherein the sodium succinate has a concentration in the basal medium of from 0.05% to 5% (w / v), wherein the phosphate source comprises sodium phosphate buffer or potassium phosphate buffer, wherein the potassium phosphate buffer has a concentration in the basal medium of from 5 to 250 mM, wherein the pH of the potassium phosphate buffer in the basal medium is from 6.7 to 7.7, wherein the ammonium source comprises ammonium chloride, and wherein the ammonium chloride has a concentration in the basal medium of from 5 to 250 mM.

[0086] In some embodiments, the sulfur source comprises sodium sulfate. In some embodiments, the concentration of the sodium sulfate in the basal medium ranges from 0.5 to 25 mM. In some embodiments, the concentration of the sodium sulfate in the basal medium ranges from 1 to 20 mM. In some embodiments, the concentration of the sodium sulfate in the basal medium ranges from 1.5 to 15 mM. In some embodiments, the concentration of the sodium sulfate in the basal medium ranges from 2 to 10 mM. In some embodiments, the concentration of the sodium sulfate in the basal medium ranges from 2.5 to 7.5 mM. In some embodiments, the concentration of the sodium sulfate in the basal medium ranges from 3.75 to 6.25 mM. In some embodiments, the concentration of the sodium sulfate in the basal medium ranges from 4.5 to 5.5 mM. In some embodiments, the concentration of the sodium sulfate in the basal medium ranges from 4.75 to 5.25 mM. In some embodiments, the concentration of the sodium sulfate in the basal medium is 5 mM. In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a basal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the basal medium comprises at least one of the following: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the sulfur source comprises sodium sulfate, and wherein the concentration of the sodium sulfate in the basal medium ranges from 0.5 to 25 mM.

[0087] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the cell-free protein synthesis system is maintained at a temperature below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowder (if present) in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the concentration of the carbon source in the cell-free protein synthesis system is from about 0 to 50 g / L, wherein the ammonium source (if present) comprises ammonium sulfate, wherein the concentration of the ammonium source in the cell-free protein synthesis system is from 2 to 20 mM, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, wherein the concentration of potassium glutamate in the cell-free protein synthesis system is from about 10 to 270 mM, wherein the concentration of potassium gluconate in the cell-free protein synthesis system is from about 10 to 250 mM, wherein the magnesium source (if present) comprises magnesium glutamate, wherein the concentration of the magnesium source in the cell-free protein synthesis system is from 0 to about 10 mM, wherein the phosphate source (if present) comprises potassium phosphate, wherein the concentration of the phosphate source in the cell-free protein synthesis system is from about 5 to about 23.5 mM, wherein the sulfur source comprises ammonium sulfate, wherein the concentration of the sulfur source in the cell-free protein synthesis system is from 2 to 20 mM, wherein the buffer comprises 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, wherein the concentration of the buffer in the cell-free protein synthesis system is from 50 to 300 mM, wherein the concentration of the cell-free cell extract in the cell-free protein synthesis system is from 10% to 70% (v / v), wherein the concentration of the DNA or RNA encoding the target protein in the cell-free protein synthesis system is from 1 to 100 μg / mL, wherein the cell-free protein synthesis system contains free amino acids endogenous to the lysed bacteria, wherein the concentration of the free amino acids in the cell-free protein synthesis system is less than 2 mM, wherein the cell-free protein synthesis system contains free nucleotides endogenous to the lysed bacteria, wherein the concentration of the free nucleotides in the cell-free protein synthesis system is less than 1.5 mM, wherein the cell-free protein synthesis system contains free NADH / NADPH endogenous to the lysed bacteria, wherein the concentration of the free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM, wherein the cell-free protein synthesis system does not contain tryptone, wherein the cell-free protein synthesis system does not contain yeast extract, wherein the basal medium comprises at least one of the following: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the carbon source is selected from D-glucose and glycerol, wherein the concentration of the D-glucose in the basal medium is from 0.1% to 2.5% (w / v), wherein the concentration of the glycerol in the basal medium is from 0.25% to 25% (w / v), wherein the concentration of the sodium succinate in the basal medium is from 0.05% to 5% (w / v), wherein the phosphate source comprises sodium phosphate buffer or potassium phosphate buffer, wherein the concentration of the potassium phosphate buffer in the basal medium is from 5 to 250 mM, wherein the pH of the potassium phosphate buffer in the basal medium is from 6.7 to 7.7, wherein the ammonium source comprises ammonium chloride, wherein the concentration of the ammonium chloride in the basal medium is from 5 to 250 mM, wherein the sulfur source comprises sodium sulfate, and wherein the concentration of the sodium sulfate in the basal medium is from 0.5 to 25 mM.

[0088] In some embodiments, the magnesium source includes magnesium sulfate heptahydrate. In some embodiments, the concentration of the magnesium sulfate heptahydrate in the basal medium ranges from 0.2 to 20 mM. In some embodiments, the concentration of the magnesium sulfate heptahydrate in the basal medium ranges from 0.3 to 19 mM. In some embodiments, the concentration of the magnesium sulfate heptahydrate in the basal medium ranges from 0.4 to 18 mM. In some embodiments, the concentration of the magnesium sulfate heptahydrate in the basal medium ranges from 0.5 to 17 mM. In some embodiments, the concentration of the magnesium sulfate heptahydrate in the basal medium ranges from 0.6 to 16 mM. In some embodiments, the concentration of the magnesium sulfate heptahydrate in the basal medium ranges from 0.7 to 15 mM. In some embodiments, the concentration of the magnesium sulfate heptahydrate in the basal medium ranges from 0.8 to 14 mM. In some embodiments, the concentration of the magnesium sulfate heptahydrate in the basal medium ranges from 0.9 to 13 mM. In some embodiments, the concentration of the magnesium sulfate heptahydrate in the basal medium ranges from 1 to 12 mM. In some embodiments, the concentration of the magnesium sulfate heptahydrate in the basal medium ranges from 1.1 to 11 mM. In some embodiments, the concentration of the magnesium sulfate heptahydrate in the basal medium ranges from 1.2 to 10 mM. In some embodiments, the concentration of the magnesium sulfate heptahydrate in the basal medium ranges from 1.3 to 9 mM. In some embodiments, the concentration of the magnesium sulfate heptahydrate in the basal medium ranges from 1.4 to 8 mM. In some embodiments, the concentration of the magnesium sulfate heptahydrate in the basal medium ranges from 1.5 to 7 mM. In some embodiments, the concentration of the magnesium sulfate heptahydrate in the basal medium ranges from 1.6 to 6 mM. In some embodiments, the concentration of the magnesium sulfate heptahydrate in the basal medium ranges from 1.7 to 5 mM. In some embodiments, the concentration of the magnesium sulfate heptahydrate in the basal medium ranges from 1.8 to 4 mM. In some embodiments, the concentration of the magnesium sulfate heptahydrate in the basal medium ranges from 1.8 to 3 mM. In some embodiments, the concentration of the magnesium sulfate heptahydrate in the basal medium ranges from 1.9 to 2.5 mM. In some embodiments, the concentration of the magnesium sulfate heptahydrate in the basal medium ranges from 1.9 to 2.25 mM. In some embodiments, the concentration of the magnesium sulfate heptahydrate in the basal medium ranges from 1.9 to 2.1 mM. In some embodiments, the concentration of the magnesium sulfate heptahydrate in the basal medium is 2 mM.In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the minimal medium comprises at least one of the following: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the magnesium source comprises magnesium sulfate heptahydrate, and wherein the concentration of the magnesium sulfate heptahydrate in the minimal medium is from 0.2 to 20 mM.

[0089] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the cell-free protein synthesis system is maintained at a temperature below about 30°C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; (g) a sulfur source; and (h) a buffer, wherein the molecular crowder is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowder (if present) in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the concentration of the carbon source in the cell-free protein synthesis system is from about 0 to 50 g / L, wherein the ammonium source (if present) comprises ammonium sulfate, wherein the concentration of the ammonium source in the cell-free protein synthesis system is from 2 to 20 mM, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, wherein the concentration of potassium glutamate in the cell-free protein synthesis system is from about 10 to 270 mM, wherein the concentration of potassium gluconate in the cell-free protein synthesis system is from about 10 to 250 mM, wherein the magnesium source (if present) comprises magnesium glutamate, wherein the concentration of the magnesium source in the cell-free protein synthesis system is from 0 to about 10 mM, wherein the phosphate source (if present) comprises potassium phosphate, wherein the concentration of the phosphate source in the cell-free protein synthesis system is from about 5 to about 23.5 mM, wherein the sulfur source comprises ammonium sulfate, wherein the concentration of the sulfur source in the cell-free protein synthesis system is from 2 to 20 mM, wherein the buffer (if present) comprises 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, wherein the concentration of the buffer in the cell-free protein synthesis system is from 50 to 300 mM, wherein the concentration of the cell-free cell extract in the cell-free protein synthesis system is from 10% to 70% (v / v), wherein the concentration of the DNA or RNA encoding the target protein in the cell-free protein synthesis system is from 1 to 100 μg / mL, wherein the cell-free protein synthesis system contains free amino acids endogenous to the lysed bacteria, wherein the concentration of the free amino acids in the cell-free protein synthesis system is less than 2 mM, wherein the cell-free protein synthesis system contains free nucleotides endogenous to the lysed bacteria, wherein the concentration of the free nucleotides in the cell-free protein synthesis system is less than 1.5 mM, wherein the cell-free protein synthesis system contains free NADH / NADPH endogenous to the lysed bacteria, wherein the concentration of the free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM, wherein the cell-free protein synthesis system does not contain tryptone, wherein the cell-free protein synthesis system does not contain yeast extract, wherein the basal medium comprises at least one of the following: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the carbon source is selected from D-glucose and glycerol, wherein the concentration of the D-glucose in the basal medium is from 0.1% to 2.5% (w / v), wherein the concentration of the glycerol in the basal medium is from 0.25% to 25% (w / v), wherein the concentration of the sodium succinate in the basal medium is from 0.05% to 5% (w / v), wherein the phosphate source comprises sodium phosphate buffer or potassium phosphate buffer, wherein the concentration of the potassium phosphate buffer in the basal medium is from 5 to 250 mM, wherein the pH of the potassium phosphate buffer in the basal medium is from 6.7 to 7.7, wherein the ammonium source comprises ammonium chloride, wherein the concentration of the ammonium chloride in the basal medium is from 5 to 250 mM, wherein the sulfur source comprises sodium sulfate, and wherein the sulfur source comprises sodium sulfate, wherein the concentration of the sodium sulfate in the basal medium is from 0.5 to 25 mM, wherein the magnesium source comprises magnesium sulfate heptahydrate, and wherein the concentration of the magnesium sulfate heptahydrate in the basal medium is from 0.2 to 20 mM.

[0090] In some embodiments, the trace mineral source is selected from an iron source, a copper source, a nickel source, a zinc source, a molybdenum source, a boron source, and a manganese source. In some embodiments, the iron source includes iron citrate. In some embodiments, the concentration of the iron citrate in the basal medium ranges from 10 to 1,000 μM. In some embodiments, the concentration of the iron citrate in the basal medium ranges from 20 to 900 μM. In some embodiments, the concentration of the iron citrate in the basal medium ranges from 30 to 800 μM. In some embodiments, the concentration of the iron citrate in the basal medium ranges from 40 to 700 μM. In some embodiments, the concentration of the iron citrate in the basal medium ranges from 50 to 600 μM. In some embodiments, the concentration of the iron citrate in the basal medium ranges from 60 to 500 μM. In some embodiments, the concentration of the iron citrate in the basal medium ranges from 70 to 400 μM. In some embodiments, the concentration of the iron citrate in the basal medium ranges from 80 to 300 μM. In some embodiments, the concentration of the iron citrate in the basal medium ranges from 90 to 200 μM. In some embodiments, the concentration of the iron citrate in the basal medium ranges from 90 to 150 μM. In some embodiments, the concentration of the iron citrate in the basal medium ranges from 95 to 125 μM. In some embodiments, the concentration of the iron citrate in the basal medium ranges from 95 to 105 μM. In some embodiments, the concentration of the iron citrate in the basal medium is 100 μM. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a basal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless the free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the basal medium comprises at least one of the following: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the trace mineral source is selected from an iron source, a copper source, a nickel source, a zinc source, a molybdenum source, a boron source, and a manganese source, wherein the iron source includes iron citrate, and wherein the concentration of the iron citrate in the basal medium ranges from 10 to 1,000 μM.

[0091] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the cell-free protein synthesis system is maintained at a temperature below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowder (if present) in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the concentration of the carbon source in the cell-free protein synthesis system is from about 0 to 50 g / L, wherein the ammonium source (if present) comprises ammonium sulfate, wherein the concentration of the ammonium source in the cell-free protein synthesis system is from 2 to 20 mM, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, wherein the concentration of potassium glutamate in the cell-free protein synthesis system is from about 10 to 270 mM, wherein the concentration of potassium gluconate in the cell-free protein synthesis system is from about 10 to 250 mM, wherein the magnesium source (if present) comprises magnesium glutamate, wherein the concentration of the magnesium source in the cell-free protein synthesis system is from 0 to about 10 mM, wherein the phosphate source (if present) comprises potassium phosphate, wherein the concentration of the phosphate source in the cell-free protein synthesis system is from about 5 to about 23.5 mM, wherein the sulfur source (if present) comprises ammonium sulfate, wherein the concentration of the sulfur source in the cell-free protein synthesis system is from 2 to 20 mM, wherein the buffer (if present) comprises 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, wherein the concentration of the buffer in the cell-free protein synthesis system is from 50 to 300 mM, wherein the concentration of the cell-free cell extract in the cell-free protein synthesis system is from 10% to 70% (v / v), wherein the DNA or RNA encoding the protein of interest is at a concentration of from 1 to 100 μg / mL in the cell-free protein synthesis system, wherein the cell-free protein synthesis system contains free amino acids endogenous to the lysed bacteria, wherein the concentration of free amino acids in the cell-free protein synthesis system is less than 2 mM, wherein the cell-free protein synthesis system contains free nucleotides endogenous to the lysed bacteria, wherein the concentration of free nucleotides in the cell-free protein synthesis system is less than 1.5 mM, wherein the cell-free protein synthesis system contains free NADH / NADPH endogenous to the lysed bacteria, wherein the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM, wherein the cell-free protein synthesis system does not contain tryptone, wherein the cell-free protein synthesis system does not contain yeast extract, wherein the basal medium comprises at least one of the following: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the carbon source is selected from D-glucose and glycerol, wherein the concentration of D-glucose in the basal medium is from 0.1% to 2.5% (w / v), wherein the concentration of glycerol in the basal medium is from 0.25% to 25% (w / v), wherein the concentration of sodium succinate in the basal medium is from 0.05% to 5% (w / v), wherein the phosphate source comprises sodium phosphate buffer or potassium phosphate buffer, wherein the concentration of potassium phosphate buffer in the basal medium is from 5 to 250 mM, wherein the pH of the potassium phosphate buffer in the basal medium is from 6.7 to 7.7, wherein the ammonium source comprises ammonium chloride, wherein the concentration of ammonium chloride in the basal medium is from 5 to 250 mM, wherein the sulfur source comprises sodium sulfate, and wherein the sulfur source comprises sodium sulfate, wherein the concentration of sodium sulfate in the basal medium is from 0.5 to 25 mM, wherein the magnesium source comprises magnesium sulfate heptahydrate, wherein the concentration of magnesium sulfate heptahydrate in the basal medium is from 0.2 to 20 mM, wherein the trace mineral source is selected from iron source, copper source, nickel source, zinc source, molybdenum source, boron source and manganese source, wherein the iron source comprises ferric citrate, and wherein the concentration of ferric citrate in the basal medium is from 10 to 1,000 μM. Bacterial culture

[0092] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, and wherein the bacteria are Escherichia coli (E. coli), Bacillus subtilis (B. subtilis), or Vibrio natriegens (V. natriegens).

[0093] In some embodiments, the bacteria are grown in any minimal medium. In some embodiments, the bacteria are grown in lysogeny broth (LB) medium. In some embodiments, the bacteria are pelleted (i.e., the supernatant is removed and the bacteria are spun down), and then the pellet is resuspended in the minimal medium at 4% (v / v), optionally wherein the minimal medium further comprises about 12.5 μg / mL chloramphenicol and 50 μg / mL isopropyl β-D-1-thiogalactopyranoside (IPTG).

[0094] In some embodiments, the bacteria are cultured in the minimal medium at 37 °C and monitored until an OD600 of 7 to 10 is reached. In some embodiments, the bacteria are pelleted and the supernatant is discarded. In some embodiments, the bacteria are washed once with a buffer at a ratio of 20 mL buffer / gram of bacteria, the buffer containing 10 mM Tris-acetate (pH 7.9), 14 mM magnesium glutamate, and 60 mM potassium glutamate. In some embodiments, the bacteria are pelleted and resuspended in the buffer at a ratio of 1.1 mL buffer / gram of bacteria to produce a cell-buffer suspension. In some embodiments, the cell-buffer suspension is immersed in an ice-water bath to prepare the cell-free cell extract. Cell-free cell extract

[0095] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, and wherein the cell-free cell extract is prepared by preparing a suspension in which each gram of the bacteria is suspended in 0.1 mL to 6.1 mL of suspension buffer. In some embodiments, the cell-free cell extract is prepared by preparing a suspension in which each gram of the bacteria is suspended in 0.2 mL to 5.6 mL of suspension buffer. In some embodiments, the cell-free cell extract is prepared by preparing a suspension in which each gram of the bacteria is suspended in 0.3 mL to 5.1 mL of suspension buffer. In some embodiments, the cell-free cell extract is prepared by preparing a suspension in which each gram of the bacteria is suspended in 0.4 mL to 4.6 mL of suspension buffer. In some embodiments, the cell-free cell extract is prepared by preparing a suspension in which each gram of the bacteria is suspended in 0.5 mL to 4.1 mL of suspension buffer. In some embodiments, the cell-free cell extract is prepared by preparing a suspension in which each gram of the bacteria is suspended in 0.6 mL to 3.6 mL of suspension buffer. In some embodiments, the cell-free cell extract is prepared by preparing a suspension in which each gram of the bacteria is suspended in 0.7 mL to 3.1 mL of suspension buffer. In some embodiments, the cell-free cell extract is prepared by preparing a suspension in which each gram of the bacteria is suspended in 0.8 mL to 2.6 mL of suspension buffer. In some embodiments, the cell-free cell extract is prepared by preparing a suspension in which each gram of the bacteria is suspended in 0.9 mL to 2.1 mL of suspension buffer. In some embodiments, the cell-free cell extract is prepared by preparing a suspension in which each gram of the bacteria is suspended in 1 mL to 1.6 mL of suspension buffer. In some embodiments, the cell-free cell extract is prepared by preparing a suspension in which each gram of the bacteria is suspended in 1.05 mL to 1.35 mL of suspension buffer.In some embodiments, the cell-free cell extract is prepared by preparing a suspension in which each gram of these bacteria is suspended in 1.05 mL to 1.2 mL of suspension buffer.

[0096] In some embodiments, the cell-free cell extract is prepared by preparing a suspension in which each gram of these bacteria is suspended in 1.1 mL of suspension buffer. In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the cell-free protein synthesis system is maintained at a temperature below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the molecular crowder (if present) is present in the cell-free protein synthesis system at a concentration of from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the carbon source (if present) is present in the cell-free protein synthesis system at a concentration of from about 0 to 50 g / L, wherein the ammonium source (if present) comprises ammonium sulfate, wherein the ammonium source is present in the cell-free protein synthesis system at a concentration of from 2 to 20 mM, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, wherein potassium glutamate is present in the cell-free protein synthesis system at a concentration of from about 10 to 270 mM, wherein potassium gluconate is present in the cell-free protein synthesis system at a concentration of from about 10 to 250 mM, wherein the magnesium source (if present) comprises magnesium glutamate, wherein the magnesium source is present in the cell-free protein synthesis system at a concentration of from 0 to about 10 mM, wherein the phosphate source comprises potassium phosphate, wherein the phosphate source is present in the cell-free protein synthesis system at a concentration of from about 5 to about 23.5 mM, wherein the sulfur source includes ammonium sulfate, wherein the concentration of the sulfur source in the cell-free protein synthesis system is from 2 to 20 mM, wherein the buffer (if present) includes 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, wherein the concentration of the buffer in the cell-free protein synthesis system is from 50 to 300 mM, wherein the concentration of the cell-free cell extract in the cell-free protein synthesis system is from 10% to 70% (v / v), wherein the DNA or RNA encoding the protein of interest in the cell-free protein synthesis system is from 1 to 100 μg / mL, wherein the cell-free protein synthesis system contains free amino acids endogenous to these lysed bacteria, wherein the concentration of free amino acids in the cell-free protein synthesis system is less than 2 mM, wherein the cell-free protein synthesis system contains free nucleotides endogenous to these lysed bacteria, wherein the concentration of free nucleotides in the cell-free protein synthesis system is less than 1.5 mM, wherein the cell-free protein synthesis system contains free NADH / NADPH endogenous to these lysed bacteria, wherein the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM, wherein the cell-free protein synthesis system does not contain tryptone, wherein the cell-free protein synthesis system does not contain yeast extract, wherein the basal medium contains at least one of the following: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the carbon source is selected from D-glucose and glycerol, wherein the concentration of D-glucose in the basal medium is from 0.1% to 2.5% (w / v), wherein the concentration of glycerol in the basal medium is from 0.25% to 25% (w / v), wherein the concentration of sodium succinate in the basal medium is from 0.05% to 5% (w / v), wherein the phosphate source includes sodium phosphate buffer or potassium phosphate buffer, wherein the concentration of the potassium phosphate buffer in the basal medium is from 5 to 250 mM, wherein the pH of the potassium phosphate buffer in the basal medium is from 6.7 to 7.7, wherein the ammonium source includes ammonium chloride, wherein the concentration of ammonium chloride in the basal medium is from 5 to 250 mM, wherein the sulfur source includes sodium sulfate, and wherein the sulfur source includes sodium sulfate, wherein the concentration of sodium sulfate in the basal medium is from 0.5 to 25 mM, wherein the magnesium source includes magnesium sulfate heptahydrate, wherein the concentration of magnesium sulfate heptahydrate in the basal medium is from 0.2 to 20 mM, wherein the trace mineral source is selected from iron source, copper source, nickel source, zinc source, molybdenum source, boron source and manganese source, wherein the iron source includes ferric citrate, wherein the concentration of ferric citrate in the basal medium is from 10 to 1,000 μM, and wherein the cell-free cell extract is prepared by preparing such a suspension in which each gram of these bacteria is suspended in 0.1 mL to 6.In 1 mL of suspension buffer. In some embodiments, the cell-free cell extract is prepared by lysing the bacteria in the suspension. In some embodiments, the lysis includes sonication. In some embodiments, sonication is performed using a 10-second on, 10-second off cycle and 50% amplitude. In some embodiments, sonication is performed until the input energy reaches about 3,500 joules. In some embodiments, the bacterial lysate is centrifuged. In some embodiments, the bacterial lysate is centrifuged at 12,000 RCF for 15 minutes at 4°C. In some embodiments, the supernatant is collected and immediately frozen at -80°C. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the cell-free cell extract is prepared by preparing such a suspension in which each gram of the bacteria is suspended in 0.1 mL to 6.1 mL of suspension buffer, and wherein the cell-free cell extract is prepared by lysing the bacteria in the suspension. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the cell-free cell extract is prepared by preparing such a suspension in which each gram of the bacteria is suspended in 0.1 mL to 6.1 mL of suspension buffer, wherein the cell-free cell extract is prepared by lysing the bacteria in the suspension, and wherein the lysis includes sonication.

[0097] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the cell-free protein synthesis system is maintained at a temperature below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowder (if present) in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the concentration of the carbon source (if present) in the cell-free protein synthesis system is from about 0 to 50 g / L, wherein the ammonium source (if present) comprises ammonium sulfate, wherein the concentration of the ammonium source (if present) in the cell-free protein synthesis system is from 2 to 20 mM, wherein the potassium source is selected from potassium glutamate and potassium gluconate, wherein the concentration of potassium glutamate in the cell-free protein synthesis system is from about 10 to 270 mM, wherein the concentration of potassium gluconate in the cell-free protein synthesis system is from about 10 to 250 mM, wherein the magnesium source (if present) comprises magnesium glutamate, wherein the concentration of the magnesium source in the cell-free protein synthesis system is from 0 to about 10 mM, wherein the phosphate source (if present) comprises potassium phosphate, wherein the concentration of the phosphate source in the cell-free protein synthesis system is from about 5 to about 23.5 mM, wherein the sulfur source includes ammonium sulfate, wherein the concentration of the sulfur source in the cell-free protein synthesis system is from 2 to 20 mM, wherein the buffer (if present) includes 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, wherein the concentration of the buffer in the cell-free protein synthesis system is from 50 to 300 mM, wherein the concentration of the cell-free cell extract in the cell-free protein synthesis system is from 10% to 70% (v / v), wherein the DNA or RNA encoding the target protein has a concentration in the cell-free protein synthesis system of from 1 to 100 μg / mL, wherein the cell-free protein synthesis system contains free amino acids endogenous to these lysed bacteria, wherein the free amino acids have a concentration in the cell-free protein synthesis system of less than 2 mM, wherein the cell-free protein synthesis system contains free nucleotides endogenous to these lysed bacteria, wherein the free nucleotides have a concentration in the cell-free protein synthesis system of less than 1.5 mM, wherein the cell-free protein synthesis system contains free NADH / NADPH endogenous to these lysed bacteria, wherein the free NADH / NADPH has a concentration in the cell-free protein synthesis system of less than 0.4 mM, wherein the cell-free protein synthesis system does not contain tryptone, wherein the cell-free protein synthesis system does not contain yeast extract, wherein the basal medium contains at least one of the following: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the carbon source is selected from D-glucose and glycerol, wherein the D-glucose has a concentration in the basal medium of from 0.1% to 2.5% (w / v), wherein the glycerol has a concentration in the basal medium of from 0.25% to 25% (w / v), wherein the sodium succinate has a concentration in the basal medium of from 0.05% to 5% (w / v), wherein the phosphate source includes sodium phosphate buffer or potassium phosphate buffer, wherein the potassium phosphate buffer has a concentration in the basal medium of from 5 to 250 mM, wherein the pH of the potassium phosphate buffer in the basal medium is from 6.7 to 7.7, wherein the ammonium source includes ammonium chloride, wherein the ammonium chloride has a concentration in the basal medium of from 5 to 250 mM, wherein the sulfur source includes sodium sulfate, and wherein the sulfur source includes sodium sulfate, wherein the sodium sulfate has a concentration in the basal medium of from 0.5 to 25 mM, wherein the magnesium source includes magnesium sulfate heptahydrate, wherein the magnesium sulfate heptahydrate has a concentration in the basal medium of from 0.2 to 20 mM, wherein the trace mineral source is selected from an iron source, a copper source, a nickel source, a zinc source, a molybdenum source, a boron source, and a manganese source, wherein the iron source includes ferric citrate, wherein the ferric citrate has a concentration in the basal medium of from 10 to 1,000 μM, wherein the cell-free cell extract is prepared by preparing such a suspension in which each gram of these bacteria is suspended in 0.1 mL to 6.In 1 mL of suspension buffer, wherein the cell-free cell extract is prepared by lysing bacteria in the suspension, and wherein the lysis comprises sonication.

[0098] In some embodiments, the suspension buffer comprises one or more of the following: (a) Tris-acetate; (b) magnesium glutamate; and (c) potassium glutamate. In some embodiments, the suspension buffer comprises (a) Tris-acetate; (b) magnesium glutamate; and (c) potassium glutamate. In some embodiments, provided herein is a cell-free protein synthesis system comprising: (a) a cell-free cell extract comprising a supernatant collected from lysed bacteria grown in minimal medium; and (b) a reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the cell-free cell extract is prepared by preparing such a suspension in which each gram of the bacteria is suspended in 0.1 mL to 6.1 mL of suspension buffer, and wherein the suspension buffer comprises one or more of the following: (a) Tris-acetate; (b) magnesium glutamate; and (c) potassium glutamate.

[0099] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the cell-free protein synthesis system is maintained at a temperature below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowder in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the concentration of the carbon source in the cell-free protein synthesis system is from about 0 to 50 g / L, wherein the ammonium source (if present) comprises ammonium sulfate, wherein the concentration of the ammonium source in the cell-free protein synthesis system is from 2 to 20 mM, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, wherein the concentration of potassium glutamate in the cell-free protein synthesis system is from about 10 to 270 mM, wherein the concentration of potassium gluconate in the cell-free protein synthesis system is from about 10 to 250 mM, wherein the magnesium source (if present) comprises magnesium glutamate, wherein the concentration of the magnesium source in the cell-free protein synthesis system is from 0 to about 10 mM, wherein the phosphate source (if present) comprises potassium phosphate, wherein the concentration of the phosphate source in the cell-free protein synthesis system is from about 5 to about 23.5 mM, wherein the sulfur source (if present) comprises ammonium sulfate, wherein the concentration of the sulfur source in the cell-free protein synthesis system is from 2 to 20 mM, wherein the buffer comprises 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, wherein the concentration of the buffer in the cell-free protein synthesis system is from 50 to 300 mM, wherein the concentration of the cell-free cell extract in the cell-free protein synthesis system is from 10% to 70% (v / v), wherein the DNA or RNA encoding the protein of interest is at a concentration of from 1 to 100 μg / mL in the cell-free protein synthesis system, wherein the cell-free protein synthesis system contains free amino acids endogenous to the lysed bacteria, wherein the concentration of the free amino acids in the cell-free protein synthesis system is less than 2 mM, wherein the cell-free protein synthesis system contains free nucleotides endogenous to the lysed bacteria, wherein the concentration of the free nucleotides in the cell-free protein synthesis system is less than 1.5 mM, wherein the cell-free protein synthesis system contains free NADH / NADPH endogenous to the lysed bacteria, wherein the concentration of the free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM, wherein the cell-free protein synthesis system does not contain tryptone, wherein the cell-free protein synthesis system does not contain yeast extract, wherein the basal medium comprises at least one of the following: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the carbon source is selected from D-glucose and glycerol, wherein the concentration of the D-glucose in the basal medium is from 0.1% to 2.5% (w / v), wherein the concentration of the glycerol in the basal medium is from 0.25% to 25% (w / v), wherein the concentration of the sodium succinate in the basal medium is from 0.05% to 5% (w / v), wherein the phosphate source comprises sodium phosphate buffer or potassium phosphate buffer, wherein the concentration of the potassium phosphate buffer in the basal medium is from 5 to 250 mM, wherein the pH of the potassium phosphate buffer in the basal medium is from 6.7 to 7.7, wherein the ammonium source comprises ammonium chloride, wherein the concentration of the ammonium chloride in the basal medium is from 5 to 250 mM, wherein the sulfur source comprises sodium sulfate, and wherein the sulfur source comprises sodium sulfate, wherein the concentration of the sodium sulfate in the basal medium is from 0.5 to 25 mM, wherein the magnesium source comprises magnesium sulfate heptahydrate, wherein the concentration of the magnesium sulfate heptahydrate in the basal medium is from 0.2 to 20 mM, wherein the trace mineral source is selected from an iron source, a copper source, a nickel source, a zinc source, a molybdenum source, a boron source, and a manganese source, wherein the iron source comprises ferric citrate, wherein the concentration of the ferric citrate in the basal medium is from 10 to 1,000 μM, wherein the cell-free cell extract is prepared by preparing such a suspension in which each gram of these bacteria is suspended in 0.1 mL to 6.In 1 mL of suspension buffer, wherein the cell-free cell extract is prepared by lysing bacteria in the suspension, wherein the lysis includes sonication, and wherein the suspension buffer contains one or more of the following: (a) Tris-acetate; (b) magnesium glutamate; and (c) potassium glutamate.

[0100] In some embodiments, the concentration of Tris-acetate in the suspension buffer is from 1 to 100 mM. In some embodiments, the concentration of Tris-acetate in the suspension buffer is from 2 to 90 mM. In some embodiments, the concentration of Tris-acetate in the suspension buffer is from 3 to 80 mM. In some embodiments, the concentration of Tris-acetate in the suspension buffer is from 4 to 70 mM. In some embodiments, the concentration of Tris-acetate in the suspension buffer is from 5 to 60 mM. In some embodiments, the concentration of Tris-acetate in the suspension buffer is from 6 to 50 mM. In some embodiments, the concentration of Tris-acetate in the suspension buffer is from 7 to 40 mM. In some embodiments, the concentration of Tris-acetate in the suspension buffer is from 8 to 30 mM. In some embodiments, the concentration of Tris-acetate in the suspension buffer is from 9 to 20 mM. In some embodiments, the concentration of Tris-acetate in the suspension buffer is from 9 to 15 mM. In some embodiments, the concentration of Tris-acetate in the suspension buffer is from 9 to 12.5 mM. In some embodiments, the concentration of Tris-acetate in the suspension buffer is from 9 to 11 mM. In some embodiments, the concentration of Tris-acetate in the suspension buffer is 10 mM. In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the cell-free cell extract is prepared by preparing such a suspension in which each gram of the bacteria is suspended in 0.1 mL to 6.1 mL of suspension buffer, wherein the suspension buffer contains one or more of the following: (a) Tris-acetate; (b) magnesium glutamate; and (c) potassium glutamate, and wherein the concentration of Tris-acetate in the suspension buffer is from 1 to 100 mM.

[0101] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the cell-free protein synthesis system is maintained at a temperature below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowder in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the concentration of the carbon source in the cell-free protein synthesis system is from about 0 to 50 g / L, wherein the ammonium source (if present) comprises ammonium sulfate, wherein the concentration of the ammonium source in the cell-free protein synthesis system is from 2 to 20 mM, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, wherein the concentration of potassium glutamate in the cell-free protein synthesis system is from about 10 to 270 mM, wherein the concentration of potassium gluconate in the cell-free protein synthesis system is from about 10 to 250 mM, wherein the magnesium source (if present) comprises magnesium glutamate, wherein the concentration of the magnesium source in the cell-free protein synthesis system is from 0 to about 10 mM, wherein the phosphate source (if present) comprises potassium phosphate, wherein the concentration of the phosphate source in the cell-free protein synthesis system is from about 5 to about 23.5 mM, wherein the sulfur source (if present) comprises ammonium sulfate, wherein the concentration of the sulfur source in the cell-free protein synthesis system is from 2 to 20 mM, wherein the buffer comprises 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, wherein the concentration of the buffer in the cell-free protein synthesis system is from 50 to 300 mM, wherein the cell-free cell extract has a concentration in the cell-free protein synthesis system of from 10% to 70% (v / v), wherein the DNA or RNA encoding the protein of interest has a concentration in the cell-free protein synthesis system of from 1 to 100 μg / mL, wherein the cell-free protein synthesis system contains free amino acids endogenous to the lysed bacteria, wherein the free amino acids have a concentration in the cell-free protein synthesis system of less than 2 mM, wherein the cell-free protein synthesis system contains free nucleotides endogenous to the lysed bacteria, wherein the free nucleotides have a concentration in the cell-free protein synthesis system of less than 1.5 mM, wherein the cell-free protein synthesis system contains free NADH / NADPH endogenous to the lysed bacteria, wherein the free NADH / NADPH has a concentration in the cell-free protein synthesis system of less than 0.4 mM, wherein the cell-free protein synthesis system does not contain tryptone, wherein the cell-free protein synthesis system does not contain yeast extract, wherein the basal medium comprises at least one of the following: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the carbon source is selected from D-glucose and glycerol, wherein the D-glucose has a concentration in the basal medium of from 0.1% to 2.5% (w / v), wherein the glycerol has a concentration in the basal medium of from 0.25% to 25% (w / v), wherein the sodium succinate has a concentration in the basal medium of from 0.05% to 5% (w / v), wherein the phosphate source comprises sodium phosphate buffer or potassium phosphate buffer, wherein the potassium phosphate buffer has a concentration in the basal medium of from 5 to 250 mM, wherein the pH of the potassium phosphate buffer in the basal medium is from 6.7 to 7.7, wherein the ammonium source comprises ammonium chloride, wherein the ammonium chloride has a concentration in the basal medium of from 5 to 250 mM, wherein the sulfur source comprises sodium sulfate, and wherein the sulfur source comprises sodium sulfate, wherein the sodium sulfate has a concentration in the basal medium of from 0.5 to 25 mM, wherein the magnesium source comprises magnesium sulfate heptahydrate, wherein the magnesium sulfate heptahydrate has a concentration in the basal medium of from 0.2 to 20 mM, wherein the trace mineral source is selected from an iron source, a copper source, a nickel source, a zinc source, a molybdenum source, a boron source, and a manganese source, wherein the iron source comprises iron citrate, wherein the iron citrate has a concentration in the basal medium of from 10 to 1,000 μM, wherein the cell-free cell extract is prepared by preparing such a suspension in which each gram of these bacteria is suspended in 0.1 mL to 6.In 1 mL of suspension buffer, wherein the cell-free cell extract is prepared by lysing bacteria in the suspension, wherein the lysis includes sonication, wherein the suspension buffer contains one or more of the following: (a) Tris-acetate; (b) magnesium glutamate; and (c) potassium glutamate, and wherein the concentration of Tris-acetate in the suspension buffer is from 1 to 100 mM.

[0102] In some embodiments, the concentration of magnesium glutamate in the suspension buffer is from 1 to 100 mM. In some embodiments, the concentration of magnesium glutamate in the suspension buffer is from 2 to 90 mM. In some embodiments, the concentration of magnesium glutamate in the suspension buffer is from 3 to 80 mM. In some embodiments, the concentration of magnesium glutamate in the suspension buffer is from 4 to 70 mM. In some embodiments, the concentration of magnesium glutamate in the suspension buffer is from 5 to 60 mM. In some embodiments, the concentration of magnesium glutamate in the suspension buffer is from 6 to 50 mM. In some embodiments, the concentration of magnesium glutamate in the suspension buffer is from 7 to 40 mM. In some embodiments, the concentration of magnesium glutamate in the suspension buffer is from 8 to 30 mM. In some embodiments, the concentration of magnesium glutamate in the suspension buffer is from 9 to 20 mM. In some embodiments, the concentration of magnesium glutamate in the suspension buffer is from 10 to 19 mM. In some embodiments, the concentration of magnesium glutamate in the suspension buffer is from 11 to 18 mM. In some embodiments, the concentration of magnesium glutamate in the suspension buffer is from 12 to 17 mM. In some embodiments, the concentration of magnesium glutamate in the suspension buffer is from 13 to 16 mM. In some embodiments, the concentration of magnesium glutamate in the suspension buffer is from 13 to 15 mM. In some embodiments, the concentration of magnesium glutamate in the suspension buffer is 14 mM. In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the cell-free cell extract is prepared by preparing such a suspension in which each gram of the bacteria is suspended in 0.1 mL to 6.1 mL of suspension buffer, wherein the suspension buffer contains one or more of the following: (a) Tris-acetate; (b) magnesium glutamate; and (c) potassium glutamate, and wherein the concentration of magnesium glutamate in the suspension buffer is from 1 to 100 mM.

[0103] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the cell-free protein synthesis system is maintained at a temperature below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowder (if present) in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the concentration of the carbon source in the cell-free protein synthesis system is from about 0 to 50 g / L, wherein the ammonium source (if present) comprises ammonium sulfate, wherein the concentration of the ammonium source in the cell-free protein synthesis system is from 2 to 20 mM, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, wherein the concentration of potassium glutamate in the cell-free protein synthesis system is from about 10 to 270 mM, wherein the concentration of potassium gluconate in the cell-free protein synthesis system is from about 10 to 250 mM, wherein the magnesium source (if present) comprises magnesium glutamate, wherein the concentration of the magnesium source in the cell-free protein synthesis system is from 0 to about 10 mM, wherein the phosphate source (if present) comprises potassium phosphate, wherein the concentration of the phosphate source in the cell-free protein synthesis system is from about 5 to about 23.5 mM, wherein the sulfur source (if present) comprises ammonium sulfate, wherein the concentration of the sulfur source in the cell-free protein synthesis system is from 2 to 20 mM, wherein the buffer comprises 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, wherein the concentration of the buffer in the cell-free protein synthesis system is from 50 to 300 mM, wherein the concentration of the cell-free cell extract in the cell-free protein synthesis system is from 10% to 70% (v / v), wherein the DNA or RNA encoding the protein of interest is at a concentration of from 1 to 100 μg / mL in the cell-free protein synthesis system, wherein the cell-free protein synthesis system contains free amino acids endogenous to the lysed bacteria, wherein the concentration of the free amino acids in the cell-free protein synthesis system is less than 2 mM, wherein the cell-free protein synthesis system contains free nucleotides endogenous to the lysed bacteria, wherein the concentration of the free nucleotides in the cell-free protein synthesis system is less than 1.5 mM, wherein the cell-free protein synthesis system contains free NADH / NADPH endogenous to the lysed bacteria, wherein the concentration of the free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM, wherein the cell-free protein synthesis system does not contain tryptone, wherein the cell-free protein synthesis system does not contain yeast extract, wherein the basal medium comprises at least one of the following: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the carbon source is selected from D-glucose and glycerol, wherein the concentration of the D-glucose in the basal medium is from 0.1% to 2.5% (w / v), wherein the concentration of the glycerol in the basal medium is from 0.25% to 25% (w / v), wherein the concentration of the sodium succinate in the basal medium is from 0.05% to 5% (w / v), wherein the phosphate source comprises sodium phosphate buffer or potassium phosphate buffer, wherein the concentration of the potassium phosphate buffer in the basal medium is from 5 to 250 mM, wherein the pH of the potassium phosphate buffer in the basal medium is from 6.7 to 7.7, wherein the ammonium source comprises ammonium chloride, wherein the concentration of the ammonium chloride in the basal medium is from 5 to 250 mM, wherein the sulfur source comprises sodium sulfate, and wherein the sulfur source comprises sodium sulfate, wherein the concentration of the sodium sulfate in the basal medium is from 0.5 to 25 mM, wherein the magnesium source comprises magnesium sulfate heptahydrate, wherein the concentration of the magnesium sulfate heptahydrate in the basal medium is from 0.2 to 20 mM, wherein the trace mineral source is selected from iron source, copper source, nickel source, zinc source, molybdenum source, boron source and manganese source, wherein the iron source comprises ferric citrate, wherein the concentration of the ferric citrate in the basal medium is from 10 to 1,000 μM, wherein the cell-free cell extract is prepared by preparing such a suspension in which each gram of these bacteria is suspended in 0.1 mL to 6.In 1 mL of suspension buffer, wherein the cell-free cell extract is prepared by lysing bacteria in the suspension, wherein the lysis includes sonication, wherein the suspension buffer contains one or more of the following: (a) Tris-acetate; (b) magnesium glutamate; and (c) potassium glutamate, wherein the concentration of Tris-acetate in the suspension buffer is from 1 to 100 mM, and wherein the concentration of magnesium glutamate in the suspension buffer is from 1 to 100 mM.

[0104] In some embodiments, the concentration of potassium glutamate in the suspension buffer ranges from 1 to 180 mM. In some embodiments, the concentration of potassium glutamate in the suspension buffer ranges from 5 to 170 mM. In some embodiments, the concentration of potassium glutamate in the suspension buffer ranges from 10 to 160 mM. In some embodiments, the concentration of potassium glutamate in the suspension buffer ranges from 15 to 150 mM. In some embodiments, the concentration of potassium glutamate in the suspension buffer ranges from 20 to 140 mM. In some embodiments, the concentration of potassium glutamate in the suspension buffer ranges from 25 to 130 mM. In some embodiments, the concentration of potassium glutamate in the suspension buffer ranges from 30 to 120 mM. In some embodiments, the concentration of potassium glutamate in the suspension buffer ranges from 35 to 110 mM. In some embodiments, the concentration of potassium glutamate in the suspension buffer ranges from 40 to 100 mM. In some embodiments, the concentration of potassium glutamate in the suspension buffer ranges from 45 to 90 mM. In some embodiments, the concentration of potassium glutamate in the suspension buffer ranges from 50 to 80 mM. In some embodiments, the concentration of potassium glutamate in the suspension buffer ranges from 55 to 70 mM. In some embodiments, the concentration of potassium glutamate in the suspension buffer ranges from 55 to 65 mM. In some embodiments, the concentration of potassium glutamate in the suspension buffer ranges from 57.5 to 62.5 mM. In some embodiments, the concentration of potassium glutamate in the suspension buffer is 60 mM. In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, and wherein the cell-free cell extract is prepared by preparing a suspension in which each gram of the bacteria is suspended in 0.1 mL to 6.1 mL of a suspension buffer that comprises one or more of the following: (a) Tris-acetate; (b) magnesium glutamate; and (c) potassium glutamate, and wherein the concentration of potassium glutamate in the suspension buffer ranges from 1 to 180 mM.

[0105] In some embodiments, provided herein is a cell-free protein synthesis system that comprises: (a) a cell-free cell extract that comprises a supernatant collected from lysed bacteria grown in a minimal medium; and (b) a reaction mixture that comprises DNA or RNA encoding a protein of interest, wherein the cell-free protein synthesis system does not comprise free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the cell-free protein synthesis system is maintained at a temperature below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder (if present) is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowder in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the concentration of the carbon source in the cell-free protein synthesis system is from about 0 to 50 g / L, wherein the ammonium source (if present) comprises ammonium sulfate, wherein the concentration of the ammonium source in the cell-free protein synthesis system is from 2 to 20 mM, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, wherein the concentration of potassium glutamate in the cell-free protein synthesis system is from about 10 to 270 mM, wherein the concentration of potassium gluconate in the cell-free protein synthesis system is from about 10 to 250 mM, wherein the magnesium source (if present) comprises magnesium glutamate, wherein the concentration of the magnesium source in the cell-free protein synthesis system is from 0 to about 10 mM, wherein the phosphate source (if present) comprises potassium phosphate, wherein the concentration of the phosphate source in the cell-free protein synthesis system is from about 5 to about 23.5 mM, wherein the sulfur source (if present) comprises ammonium sulfate, wherein the concentration of the sulfur source in the cell-free protein synthesis system is from 2 to 20 mM, wherein the buffer comprises 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, wherein the concentration of the buffer in the cell-free protein synthesis system is from 50 to 300 mM, wherein the concentration of the cell-free cell extract in the cell-free protein synthesis system is from 10% to 70% (v / v), wherein the DNA or RNA encoding the protein of interest has a concentration in the cell-free protein synthesis system of from 1 to 100 μg / mL, wherein the cell-free protein synthesis system contains free amino acids endogenous to these lysed bacteria, wherein the concentration of free amino acids in the cell-free protein synthesis system is less than 2 mM, wherein the cell-free protein synthesis system contains free nucleotides endogenous to these lysed bacteria, wherein the concentration of free nucleotides in the cell-free protein synthesis system is less than 1.5 mM, wherein the cell-free protein synthesis system contains free NADH / NADPH endogenous to these lysed bacteria, wherein the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM, wherein the cell-free protein synthesis system does not contain tryptone, wherein the cell-free protein synthesis system does not contain yeast extract, wherein the basal medium comprises at least one of the following: (a) a carbon source; (b) an ammonium source; (c) a magnesium source; (d) a phosphate source; (e) a sulfur source; (f) a trace mineral source; and (g) sodium succinate, wherein the carbon source is selected from D-glucose and glycerol, wherein the concentration of D-glucose in the basal medium is from 0.1% to 2.5% (w / v), wherein the concentration of glycerol in the basal medium is from 0.25% to 25% (w / v), wherein the concentration of sodium succinate in the basal medium is from 0.05% to 5% (w / v), wherein the phosphate source comprises sodium phosphate buffer or potassium phosphate buffer, wherein the concentration of the potassium phosphate buffer in the basal medium is from 5 to 250 mM, wherein the pH of the potassium phosphate buffer in the basal medium is from 6.7 to 7.7, wherein the ammonium source comprises ammonium chloride, wherein the concentration of ammonium chloride in the basal medium is from 5 to 250 mM, wherein the sulfur source comprises sodium sulfate, and wherein the sulfur source comprises sodium sulfate, wherein the concentration of sodium sulfate in the basal medium is from 0.5 to 25 mM, wherein the magnesium source comprises magnesium sulfate heptahydrate, wherein the concentration of magnesium sulfate heptahydrate in the basal medium is from 0.2 to 20 mM, wherein the trace mineral source is selected from iron source, copper source, nickel source, zinc source, molybdenum source, boron source, and manganese source, wherein the iron source comprises ferric citrate, wherein the concentration of ferric citrate in the basal medium is from 10 to 1,000 μM, wherein the cell-free cell extract is prepared by preparing such a suspension in which each gram of these bacteria is suspended in 0.1 mL to 6.In 1 mL of suspension buffer, wherein the cell-free cell extract is prepared by lysing bacteria in the suspension, wherein the lysis includes sonication, and wherein the suspension buffer contains one or more of the following: (a) Tris-acetate; (b) magnesium glutamate; and (c) potassium glutamate, wherein the concentration of the Tris-acetate in the suspension buffer is from 1 to 100 mM, wherein the concentration of the magnesium glutamate in the suspension buffer is from 1 to 100 mM, and wherein the concentration of the potassium glutamate in the suspension buffer is from 1 to 180 mM. Cell-free protein synthesis

[0106] In some embodiments, provided herein is a method for preparing a protein of interest, the method comprising using a cell-free protein synthesis system of any one of the embodiments disclosed herein. In some embodiments, provided herein is a method for preparing a protein of interest, the method comprising (1) incubating a reaction mixture with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the protein of interest, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless the free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30°C, wherein the system produces the protein of interest when maintained at the temperature. In some embodiments, the method comprises (3) purifying the protein of interest.

[0107] In some embodiments, provided herein is a method for preparing a protein of interest, the method comprising (1) incubating a reaction mixture with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the protein of interest, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless the free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30°C, wherein the system produces the protein of interest when maintained at the temperature, and wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5.

[0108] In some embodiments, provided herein is a method for preparing a protein of interest, the method comprising (1) incubating a reaction mixture with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the protein of interest, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless the free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30 °C, wherein the system produces the protein of interest when maintained at the temperature, and wherein the temperature is below about 30 °C.

[0109] In some embodiments, provided herein is a method for preparing a protein of interest, the method comprising (1) incubating a reaction mixture with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the protein of interest, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless the free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30 °C, wherein the system produces the protein of interest when maintained at the temperature, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, and wherein the temperature is below about 30 °C.

[0110] In some embodiments, provided herein is a method for preparing a protein of interest, the method comprising (1) incubating a reaction mixture with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the protein of interest, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless the free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30 °C, wherein the system produces the protein of interest when maintained at the temperature, and wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) a nitrogen source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer.

[0111] In some embodiments, provided herein is a method for preparing a protein of interest, the method comprising (1) incubating a reaction mixture with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the protein of interest, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless the free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30 °C, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the temperature is below about 30 °C, and wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; (g) a sulfur source; and (h) a buffer.

[0112] In some embodiments, provided herein is a method for preparing a protein of interest, the method comprising (1) incubating a reaction mixture with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the protein of interest, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless the free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30 °C, wherein the system produces the protein of interest when maintained at the temperature, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; (g) a sulfur source; and (h) a buffer, and wherein the molecular crowder is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin.

[0113] In some embodiments, provided herein is a method for preparing a target protein, the method comprising (1) incubating a reaction mixture with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the target protein, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30 °C, wherein the system produces the target protein when maintained at the temperature, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the temperature is below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowding agent; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, and wherein the molecular crowding agent is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin.

[0114] In some embodiments, provided herein is a method for preparing a target protein, the method comprising (1) incubating a reaction mixture with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the target protein, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowding agent; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, and wherein the concentration of the molecular crowding agent in the cell-free protein synthesis system is from about 0 to 40 g / L.

[0115] In some embodiments, provided herein is a method for preparing a target protein, the method comprising (1) incubating a reaction mixture with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the target protein, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless the free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30 °C, wherein the system produces the target protein when maintained at the temperature, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the temperature is below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowding agent; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowding agent is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, and wherein the concentration of the molecular crowding agent in the cell-free protein synthesis system is from about 0 to 40 g / L.

[0116] In some embodiments, provided herein is a method for preparing a target protein, the method comprising (1) incubating a reaction mixture with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the target protein, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless the free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30 °C, wherein the system produces the target protein when maintained at the temperature, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowding agent; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, and wherein the carbon source is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose.

[0117] In some embodiments, provided herein is a method for preparing a protein of interest, the method comprising (1) incubating a reaction mixture with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the protein of interest, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless the free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30°C, wherein the system produces the protein of interest when maintained at the temperature, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the temperature is below about 30°C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowding agent; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowding agent is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowding agent in the cell-free protein synthesis system is from about 0 to 40 g / L, and wherein the carbon source is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose.

[0118] In some embodiments, provided herein is a method for preparing a protein of interest, the method comprising (1) incubating a reaction mixture with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the protein of interest, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless the free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30°C, wherein the system produces the protein of interest when maintained at the temperature, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowding agent; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, and wherein the concentration of the carbon source in the cell-free protein synthesis system is from about 0 to 50 g / L.

[0119] In some embodiments, provided herein is a method for preparing a protein of interest, the method comprising (1) incubating a reaction mixture with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the protein of interest, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30 °C, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the temperature is below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowder in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, and wherein the concentration of the carbon source in the cell-free protein synthesis system is from about 0 to 50 g / L.

[0120] In some embodiments, provided herein is a method for preparing a protein of interest, the method comprising (1) incubating a reaction mixture with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the protein of interest, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30 °C, wherein the system produces the protein of interest when maintained at the temperature, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, and wherein the concentration of the ammonium source in the cell-free protein synthesis system is from 2 to 20 mM.

[0121] In some embodiments, provided herein is a method for preparing a target protein, the method comprising (1) incubating a reaction mixture with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the target protein, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30 °C, wherein the system produces the target protein when maintained at this temperature, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the temperature is below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowder (if present) in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the concentration of the carbon source in the cell-free protein synthesis system is from about 0 to 50 g / L, and wherein the concentration of the ammonium source (if present) in the cell-free protein synthesis system is from 2 to 20 mM.

[0122] In some embodiments, provided herein is a method for preparing a target protein, the method comprising (1) incubating a reaction mixture with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the target protein, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30 °C, wherein the system produces the target protein when maintained at this temperature, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, and wherein the potassium source is selected from potassium glutamate and potassium gluconate.

[0123] In some embodiments, provided herein is a method for preparing a target protein, the method comprising (1) incubating a reaction mixture with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the target protein, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30 °C, wherein the system produces the target protein when maintained at the temperature, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the temperature is below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; (g) a sulfur source; and (h) a buffer, wherein the molecular crowder (if present) is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowder (if present) in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the concentration of the carbon source in the cell-free protein synthesis system is from about 0 to 50 g / L, wherein the concentration of the ammonium source (if present) in the cell-free protein synthesis system is from 2 to 20 mM, and wherein the potassium source is selected from potassium glutamate and potassium gluconate.

[0124] In some embodiments, provided herein is a method for preparing a target protein, the method comprising (1) incubating a reaction mixture, which contains DNA or RNA encoding the target protein, with a cell-free cell extract, which contains a supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30 °C, wherein the system produces the target protein when maintained at this temperature, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowding agent; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, and wherein the concentration of potassium glutamate in the cell-free protein synthesis system is from about 10 to 270 mM.

[0125] In some embodiments, provided herein is a method for preparing a target protein, the method comprising (1) incubating a reaction mixture with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the target protein, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30 °C, wherein the system produces the target protein when maintained at the temperature, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the temperature is below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowding agent; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowding agent is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowding agent in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the concentration of the carbon source in the cell-free protein synthesis system is from about 0 to 50 g / L, wherein the ammonium source (if present) has a concentration in the cell-free protein synthesis system of from 2 to 20 mM, wherein the potassium source is selected from potassium glutamate and potassium gluconate, and wherein the potassium glutamate has a concentration in the cell-free protein synthesis system of from about 10 to 270 mM.

[0126] In some embodiments, provided herein is a method for preparing a target protein, the method comprising (1) incubating a reaction mixture together with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the target protein, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30°C, wherein the system produces the target protein when maintained at this temperature, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowding agent; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; (g) a sulfur source; and (h) a buffer, and wherein the concentration of potassium gluconate in the cell-free protein synthesis system is from about 10 to 250 mM.

[0127] In some embodiments, provided herein is a method for preparing a target protein, the method comprising (1) incubating a reaction mixture together with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the target protein, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 3**0**°C, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the temperature is below about 3**0**°C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowding agent; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; (g) a sulfur source; and (h) a buffer, wherein the molecular crowding agent (if present) is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowding agent in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the concentration of the carbon source in the cell-free protein synthesis system is from about 0 to 50 g / L, wherein the ammonium source (if present) has a concentration in the cell-free protein synthesis system of from 2 to 20 mM, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, wherein the concentration of potassium glutamate in the cell-free protein synthesis system is from about 10 to 270 mM, and wherein the concentration of potassium gluconate in the cell-free protein synthesis system is from about 10 to 250 mM.

[0128] In some embodiments, provided herein is a method for preparing a target protein, the method comprising (1) incubating a reaction mixture with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the target protein, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30 °C, wherein the system produces the target protein when maintained at the temperature, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowding agent; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; (g) a sulfur source; and (h) a buffer, and wherein the concentration of the magnesium source in the cell-free protein synthesis system is from 0 to about 10 mM.

[0129] In some embodiments, provided herein is a method for producing a target protein, the method comprising ((1) incubating a reaction mixture with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the target protein, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless the free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30°C, wherein the system produces the target protein when maintained at the temperature, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the temperature is below about 30°C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; (g) a sulfur source; and (h) a buffer, wherein the molecular crowder (if present) is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowder in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the concentration of the carbon source in the cell-free protein synthesis system is from about 0 to 50 g / L, wherein the ammonium source (if present) has a concentration in the cell-free protein synthesis system of from 2 to 20 mM, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, wherein the concentration of potassium glutamate in the cell-free protein synthesis system is from about 10 to 270 mM, wherein the concentration of potassium gluconate in the cell-free protein synthesis system is from about 10 to 250 mM, and wherein the magnesium source (if present) has a concentration in the cell-free protein synthesis system of from 0 to about 10 mM.

[0130] In some embodiments, provided herein is a method for preparing a target protein, the method comprising (1) incubating a reaction mixture with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the target protein, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30 °C, wherein the system produces the target protein when maintained at the temperature, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; (g) a sulfur source; and (h) a buffer, and wherein the concentration of the phosphate source in the cell-free protein synthesis system is from about 5 to about 23.5 mM.

[0131] In some embodiments, provided herein is a method for preparing a target protein, the method comprising (1) incubating a reaction mixture with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the target protein, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30 °C, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the temperature is below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowding agent; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowding agent is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowding agent (if present) in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the concentration of the carbon source in the cell-free protein synthesis system is from about 0 to 50 g / L, wherein the concentration of the ammonium source (if present) in the cell-free protein synthesis system is from 2 to 20 mM, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, wherein the concentration of potassium glutamate in the cell-free protein synthesis system is from about 10 to 270 mM, wherein the concentration of potassium gluconate in the cell-free protein synthesis system is from about 10 to 250 mM, wherein the concentration of the magnesium source (if present) in the cell-free protein synthesis system is from 0 to about 10 mM, and wherein the concentration of the phosphate source (if present) in the cell-free protein synthesis system is from about 5 to about 23.5 mM.

[0132] In some embodiments, provided herein is a method for producing a target protein, the method comprising (1) incubating a reaction mixture with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the target protein, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30°C, wherein the system produces the target protein when maintained at the temperature, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowding agent; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, and wherein the concentration of the sulfur source in the cell-free protein synthesis system is from 2 to 20 mM.

[0133] In some embodiments, provided herein is a method for preparing a target protein, the method comprising (1) incubating a reaction mixture with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the target protein, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30°C, wherein the system produces the target protein when maintained at the temperature, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the temperature is below about 30°C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder (if present) is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowder in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the concentration of the carbon source in the cell-free protein synthesis system is from about 0 to 50 g / L, wherein the ammonium source (if present) has a concentration in the cell-free protein synthesis system of from 2 to 20 mM, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, wherein the concentration of potassium glutamate in the cell-free protein synthesis system is from about 10 to 270 mM, wherein the concentration of potassium gluconate in the cell-free protein synthesis system is from about 10 to 250 mM, wherein the magnesium source (if present) has a concentration in the cell-free protein synthesis system of from 0 to about 10 mM, wherein the phosphate source (if present) has a concentration in the cell-free protein synthesis system of from about 5 to about 23.5 mM, and wherein the sulfur source (if present) has a concentration in the cell-free protein synthesis system of from 2 to 20 mM.

[0134] In some embodiments, provided herein is a method for preparing a target protein, the method comprising (1) incubating a reaction mixture with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the target protein, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30 °C, wherein the system produces the target protein when maintained at the temperature, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowding agent; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; (g) a sulfur source; and (h) a buffer, and wherein the concentration of the buffer in the cell-free protein synthesis system is from 50 to 300 mM.

[0135] In some embodiments, provided herein is a method for preparing a target protein, the method comprising (1) incubating a reaction mixture with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the target protein, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30 °C, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the temperature is below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowder is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the molecular crowder (if present) is at a concentration of from about 0 to 40 g / L in the cell-free protein synthesis system, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the carbon source is at a concentration of from about 0 to 50 g / L in the cell-free protein synthesis system, wherein the ammonium source (if present) is at a concentration of from 2 to 20 mM in the cell-free protein synthesis system, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, wherein the potassium glutamate is at a concentration of from about 10 to 270 mM in the cell-free protein synthesis system, wherein the potassium gluconate is at a concentration of from about 10 to 250 mM in the cell-free protein synthesis system, wherein the magnesium source (if present) is at a concentration of from 0 to about 10 mM in the cell-free protein synthesis system, wherein the phosphate source (if present) is at a concentration of from about 5 to about 23.5 mM in the cell-free protein synthesis system, wherein the sulfur source (if present) is at a concentration of from 2 to 20 mM in the cell-free protein synthesis system, and wherein the buffer is at a concentration of from 50 to 300 mM in the cell-free protein synthesis system.

[0136] In some embodiments, provided herein is a method for preparing a target protein, the method comprising (1) incubating a reaction mixture, which contains DNA or RNA encoding the target protein, with a cell-free cell extract, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30 °C, wherein the system produces the target protein when maintained at the temperature, and wherein the concentration of the cell-free cell extract in the cell-free protein synthesis system is from 10% to 70% (v / v).

[0137] In some embodiments, provided herein is a method for preparing a target protein, the method comprising (1) incubating a reaction mixture with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the target protein, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30°C, wherein the system produces the target protein when maintained at this temperature, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the temperature is below about 30°C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowding agent; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowding agent (if present) is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowding agent in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the concentration of the carbon source in the cell-free protein synthesis system is from about 0 to 50 g / L, wherein the ammonium source (if present) has a concentration in the cell-free protein synthesis system of from 2 to 20 mM, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, wherein the concentration of potassium glutamate in the cell-free protein synthesis system is from about 10 to 270 mM, wherein the concentration of potassium gluconate in the cell-free protein synthesis system is from about 10 to 250 mM, wherein the magnesium source (if present) has a concentration in the cell-free protein synthesis system of from 0 to about 10 mM, wherein the phosphate source (if present) has a concentration in the cell-free protein synthesis system of from about 5 to about 23.5 mM, wherein the sulfur source (if present) has a concentration in the cell-free protein synthesis system of from 2 to 20 mM, and wherein the buffer has a concentration in the cell-free protein synthesis system of from 50 to 300 mM, and wherein the cell-free cell extract has a concentration in the cell-free protein synthesis system of from 10% to 70% (v / v).

[0138] In some embodiments, provided herein is a method for preparing a target protein, the method comprising (1) incubating a reaction mixture, which contains DNA or RNA encoding the target protein, with a cell-free cell extract, the cell-free cell extract containing a supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30°C, wherein the system produces the target protein when maintained at this temperature, and wherein the concentration of the DNA or RNA encoding the target protein in the cell-free protein synthesis system is from 1 to 100 μg / mL.

[0139] In some embodiments, provided herein is a method for preparing a target protein, the method comprising (1) incubating a reaction mixture with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the target protein, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides or free NADH / NADPH, unless these free amino acids, free nucleotides or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30 °C, wherein the system produces the target protein when maintained at the temperature, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6 or about 7.5, wherein the temperature is below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowding agent; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowding agent (if present) is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowding agent in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin and maltose, wherein the concentration of the carbon source (if present) in the cell-free protein synthesis system is from about 0 to 50 g / L, wherein the concentration of the ammonium source (if present) in the cell-free protein synthesis system is from 2 to 20 mM, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, wherein the concentration of potassium glutamate in the cell-free protein synthesis system is from about 10 to 270 mM, wherein the concentration of potassium gluconate in the cell-free protein synthesis system is from about 10 to 250 mM, wherein the concentration of the magnesium source (if present) in the cell-free protein synthesis system is from 0 to about 10 mM, wherein the concentration of the phosphate source (if present) in the cell-free protein synthesis system is from about 5 to about 23.5 mM, wherein the concentration of the sulfur source (if present) in the cell-free protein synthesis system is from 2 to 20 mM, and wherein the concentration of the buffer in the cell-free protein synthesis system is from 50 to 300 mM, wherein the concentration of the cell-free cell extract in the cell-free protein synthesis system is from 10% to 70% (v / v), and wherein the concentration of the DNA or RNA encoding the target protein in the cell-free protein synthesis system is from 1 to 100 μg / mL.

[0140] In some embodiments, provided herein is a method for preparing a target protein, the method comprising (1) incubating a reaction mixture with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the target protein, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30°C, wherein the system produces the target protein when maintained at the temperature, and wherein the concentration of free amino acids in the cell-free protein synthesis system is less than 2 mM.

[0141] In some embodiments, provided herein is a method for producing a target protein, the method comprising (1) incubating a reaction mixture with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the target protein, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30°C, wherein the system produces the target protein when maintained at this temperature, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the temperature is below about 30°C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowding agent; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowding agent (if present) is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowding agent in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the concentration of the carbon source in the cell-free protein synthesis system is from about 0 to 50 g / L, wherein the ammonium source (if present) has a concentration in the cell-free protein synthesis system of from 2 to 20 mM, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, wherein the concentration of potassium glutamate in the cell-free protein synthesis system is from about 10 to 270 mM, wherein the concentration of potassium gluconate in the cell-free protein synthesis system is from about 10 to 250 mM, wherein the magnesium source (if present) has a concentration in the cell-free protein synthesis system of from 0 to about 10 mM, wherein the phosphate source (if present) has a concentration in the cell-free protein synthesis system of from about 5 to about 23.5 mM, wherein the sulfur source (if present) has a concentration in the cell-free protein synthesis system of from 2 to 20 mM, and wherein the buffer has a concentration in the cell-free protein synthesis system of from 50 to 300 mM, wherein the cell-free cell extract has a concentration in the cell-free protein synthesis system of from 10% to 70% (v / v), wherein the DNA or RNA encoding the target protein has a concentration in the cell-free protein synthesis system of from 1 to 100 μg / mL, and wherein the concentration of free amino acids in the cell-free protein synthesis system is less than 2 mM.

[0142] In some embodiments, provided herein is a method for preparing a target protein, the method comprising (1) incubating a reaction mixture with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the target protein, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless the free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30 °C, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless the free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature at which the system produces the target protein when maintained at the temperature, and wherein the concentration of free nucleotides in the cell-free protein synthesis system is less than 1.5 mM.

[0143] In some embodiments, provided herein is a method for preparing a target protein, the method comprising (1) incubating a reaction mixture with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the target protein, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30 °C, wherein the target protein is produced when the system is maintained at this temperature, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the temperature is below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowding agent; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the molecular crowding agent (if present) is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran, and serum albumin, optionally bovine serum albumin, wherein the concentration of the molecular crowding agent in the cell-free protein synthesis system is from about 0 to 40 g / L, wherein the carbon source (if present) is selected from glucose, fructose, galactose, sucrose, lactose, gluconate, starch, maltodextrin, and maltose, wherein the concentration of the carbon source in the cell-free protein synthesis system is from about 0 to 50 g / L, wherein the concentration of the ammonium source (if present) in the cell-free protein synthesis system is from 2 to 20 mM, wherein the potassium source (if present) is selected from potassium glutamate and potassium gluconate, wherein the concentration of potassium glutamate in the cell-free protein synthesis system is from about 10 to 270 mM, wherein the concentration of potassium gluconate in the cell-free protein synthesis system is from about 10 to 250 mM, wherein the concentration of the magnesium source (if present) in the cell-free protein synthesis system is from 0 to about 10 mM, wherein the concentration of the phosphate source (if present) in the cell-free protein synthesis system is from about 5 to about 23.5 mM, wherein the concentration of the sulfur source (if present) in the cell-free protein synthesis system is from 2 to 20 mM, and wherein the concentration of the buffer in the cell-free protein synthesis system is from 50 to 300 mM, wherein the concentration of the cell-free cell extract in the cell-free protein synthesis system is from 10% to 70% (v / v), wherein the concentration of the DNA or RNA encoding the target protein in the cell-free protein synthesis system is from 1 to 100 μg / mL, wherein the concentration of free amino acids in the cell-free protein synthesis system is less than 2 mM, and wherein the concentration of free nucleotides in the cell-free protein synthesis system is less than 1.5 mM.

[0144] In some embodiments, provided herein is a method for producing a target protein, the method comprising (1) incubating a reaction mixture with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the target protein, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30° C., wherein the system produces the target protein when maintained at the temperature, and wherein the concentration of free NADH / NADPH in the cell-free protein synthesis system is less than 0.4 mM.

[0145] In some embodiments, provided herein is a method for preparing a target protein, the method comprising (1) incubating a reaction mixture with a cell-free cell extract, the reaction mixture comprising DNA or RNA encoding the target protein, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and (2) maintaining the system at a temperature below about 30 °C, wherein the system produces the target protein when maintained at the temperature, wherein the pH of the cell-free protein synthesis system is from about 7.0 to about 8.0, about 7.1 to 7.9, about 7.2 to 7.8, about 7.3 to 7.7, about 7.4 to 7.6, or about 7.5, wherein the temperature is below about 30 °C, wherein the reaction mixture further comprises at least one of the following: (a) a molecular crowder; (b) a carbon source; (c) an ammonium source; (d) a potassium source; (e) a magnesium source; (f) a phosphate source; and (g) a sulfur source; and (h) a buffer, wherein the ...

Claims

1. A cell-free protein synthesis system, the system comprising: a) A cell-free cell extract, the cell-free cell extract comprising a supernatant collected from lysed bacteria grown in a minimal medium; and b) A reaction mixture, the reaction mixture comprising DNA or RNA encoding a protein of interest, wherein the system does not contain free amino acids, free nucleotides or free NADH / NADPH, unless these free amino acids, free nucleotides or free NADH / NADPH are endogenous to these lysed bacteria.

2. The system according to claim 1, wherein i. These bacteria are Escherichia coli, Bacillus subtilis or Vibrio natriegens; and / or ii. The pH of the system is from about 7.0 to about 8.0, optionally 7.5; and / or iii. The system is maintained at a temperature from about 20°C to about 30°C, optionally about 25°C.

3. The system according to claim 1 or claim 2, wherein the reaction mixture further comprises: a) A molecular crowding agent; b) A carbon source; c) An ammonium source; d) A potassium source; e) A magnesium source; f) A phosphate source; and g) A sulfur source; and optionally h) A buffer.

4. The system according to claim 3, wherein i. The molecular crowding agent is selected from PEG 400 - 8000, maltodextrin, Ficoll 70 - 400, dextran and bovine serum albumin, optionally PEG 8000; and / or ii. The concentration of the molecular crowding agent in the system is from 0 to 40 g / L, optionally 20 g / L; and / or iii. The carbon source in the reaction mixture comprises sugars; and / or iv. The carbon source in the reaction mixture comprises maltose; and / or v. The carbon source in the reaction mixture does not comprise maltose; and / or vi. The concentration of the carbon source in the system is from 0 to 50 g / L, optionally 20 g / L; and / or vii. The ammonium source in the reaction mixture comprises ammonium sulfate; and / or viii. The concentration of the ammonium source in the system is from 2 to 20 mM, optionally 12 mM; and / or ix. The potassium source in the reaction mixture is selected from potassium glutamate and potassium gluconate; and / or x. The potassium source in the reaction mixture is potassium glutamate, and the concentration of potassium glutamate in the system is from 10 to 300 mM, optionally 20 mM; and / or xi. The potassium source in the reaction mixture is potassium gluconate, and the concentration of potassium gluconate in the system is from 10 to 250 mM, optionally 100 mM; and / or xii. The magnesium source in the reaction mixture comprises magnesium glutamate; and / or xiii. The concentration of the magnesium source in the system is from 0 to 10 mM, optionally 4 mM; and / or xiv. The phosphate source in the reaction mixture comprises potassium phosphate; and / or xv. The concentration of the phosphate source in the system is from 5 to 25 mM, optionally 13 mM; and / or xvi. The sulfur source in the reaction mixture comprises ammonium sulfate; and / or xvii. The concentration of the sulfur source in the system is from 2 to 20 mM, optionally 12 mM; and / or xviii. The system comprises the buffer in the reaction mixture, wherein the buffer comprises 3-(N-morpholino)propanesulfonic acid (MOPS) buffer; and / or xix. wherein the concentration of the buffer in the system is from 50 to 300 mM, optionally 100 mM.

5. The system according to any one of the preceding claims, wherein the basal medium comprises: a) a carbon source; b) an ammonium source; c) a magnesium source; d) a phosphate source; e) a sulfur source; and f) a trace mineral source; and optionally g) sodium succinate; wherein optionally i. the carbon source in the basal medium is selected from D-glucose and glycerol; and / or ii. the concentration of the carbon source in the basal medium is from 0.5% to 4% (w / v); and / or iii. the concentration of the glycerol in the basal medium is 2.5% (w / v); and / or iv. the ammonium source in the basal medium comprises ammonium chloride; and / or v. the concentration of the ammonium source in the basal medium is 50 mM; and / or vi. the magnesium source in the basal medium comprises magnesium sulfate heptahydrate; and / or vii. the concentration of the magnesium source in the basal medium is 2 mM; and / or viii. the phosphate source in the basal medium comprises sodium phosphate buffer or potassium phosphate buffer; and / or ix. the concentration of the phosphate source in the basal medium is from 25 mM to 150 mM; and / or x. the concentration of the phosphate source in the basal medium is 50 mM; and / or xi. the pH of the sodium phosphate buffer or the potassium phosphate buffer in the basal medium is 7.2; and / or xii. the sulfur source in the basal medium comprises sodium sulfate; and / or xiii. the concentration of the sulfur source in the basal medium is 5 mM; and / or xiv. the trace mineral source in the basal medium is selected from iron source, copper source, nickel source, zinc source, molybdenum source, boron source and manganese source; and / or xv. the iron source comprises iron citrate; and / or xvi. the concentration of the iron source in the basal medium is 100 μM; and / or xvii. the basal medium comprises sodium succinate, and the concentration of the sodium succinate in the basal medium is 0.375% (w / v).

6. A method for preparing a target protein, the method comprising using the system according to any one of the preceding claims.

7. A reaction mixture for use in a cell-free protein synthesis system, the reaction mixture comprising DNA or RNA encoding a target protein, and further comprising: a) a molecular crowding agent; b) a carbon source; c) an ammonium source; d) a potassium source; e) a magnesium source; f) a phosphate source; and g) a sulfur source; and optionally h) a buffer; and optionally i) sodium chloride wherein optionally one or more of components (a) to (h) are as defined in claim 4.

8. A method for preparing a target protein, the method comprising 1) Incubate the reaction mixture, which contains DNA or RNA encoding the protein of interest, with a cell-free cell extract that contains the supernatant collected from lysed bacteria grown in minimal medium, wherein the system does not contain free amino acids, free nucleotides, or free NADH / NADPH, unless these free amino acids, free nucleotides, or free NADH / NADPH are endogenous to the lysed bacteria; and 2) Maintain the system at a temperature below about 30 °C, wherein the protein of interest is produced when the system is maintained at this temperature.

9. The method according to claim 8, wherein the method further comprises purifying the protein of interest.

10. The method according to claim 8 or claim 9, wherein the bacteria are Escherichia coli, Bacillus subtilis, or Vibrio natriegens.

11. The method according to any one of claims 8-10, wherein the reaction mixture further comprises: a) a molecular crowding agent; b) a carbon source; c) a nitrogen source; d) a potassium source; e) a magnesium source; f) a phosphate source; and g) a sulfur source; and optionally h) a buffer; wherein optionally one or more of components (a) to (h) are as defined in claim 4.

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