Diversified bacteriophage synthesis and engineering platform

Abstract

The present disclosure provides modified bacterial cell lysate compositions and methods for using the same for cell-free bacteriophage synthesis (CFBS) to generate a broad range of bacteriophage species. Also disclosed herein are phage engineering methods for converting a lysogenic bacteriophage into a lytic bacteriophage, as well as methods for generating a genetically modified bacteriophage species that targets a broader range of bacterial host strains compared to the wild-type bacteriophage species.

Description

Atty. Dkt. No.: 136669-0119 DIVERSIFIED BACTERIOPHAGE SYNTHESIS AND ENGINEERING PLATFORM CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Appl. No. 63 / 582,431 filed September 13, 2023, the contents of which are incorporated herein by reference in their entirety for any and all purposes. TECHNICAL FIELD

[0002] The present disclosure relates to modified bacterial cell lysate compositions (e.g., mixtures of E. coli and non- E. coli lysates) and methods for using the same for cell-free bacteriophage synthesis (CFBS) to generate a broad range of bacteriophage species. Also disclosed herein are phage engineering methods for converting a lysogenic bacteriophage into a lytic bacteriophage, as well as phage engineering methods for generating a genetically modified bacteriophage species that infects and kills a broader range of bacterial strains compared to the wild-type bacteriophage species. BACKGROUND

[0003] The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

[0004] Multidrug-resistant (MDR) bacteria pose one of the greatest emerging health threats. These pathogens possess intrinsic and acquired mechanisms of resistance to most common antibiotics, leaving few treatment options for the most at-risk patients. The Centers for Disease Control reports 2.8 million MDR infections per year leading to 35,000 deaths with numbers are on the rise. Phage therapy, a resurgent anti-bacterial treatment, has the potential to cure bacterial infections resistant to small molecule antibiotics. Unlike small molecule antibiotics, phage do not harm beneficial bacterial flora that protect against future infections.

[0005] Despite a promising future, phage therapy is encumbered by logistical challenges in the manufacturing process. Once a suitable phage has been identified for a particular application, it needs to be propagated using a working host, which ideally is well characterized, non- pathogenic, and free of genomically encoded prophage to prevent potential contamination of phage preparations with lysogenic phage. Such a host is often not immediately available. Next, crude phage preparations must be purified to remove endotoxin, a component of outer membranes of Gram-negative bacteria that induces septic shock. While several organic solvent 1 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 extraction or affinity column purification methods exist to remove endotoxin, these approaches have widely varying efficiencies depending on the phage undergoing purification and typically result in significant loss of titer. Additionally, low shelf-stability can result in a significant loss of titer prior to clinical administration due to storage conditions, shipping conditions, and phage- dependent variability.

[0006] Accordingly, there is an urgent need for phage production methods that achieve enhanced yields over conventional cell-free bacteriophage synthesis (CFBS) methods. SUMMARY OF THE PRESENT TECHNOLOGY

[0007] In one aspect, the present disclosure provides a modified bacterial cell lysate composition comprising a mixture of an E. coli lysate and at least one additional bacterial cell lysate obtained from a bacterial host species that is not E. coli, wherein the cell lysate composition comprises an effective amount of transcription / translation (TXTL) machinery that is configured to synthesize at least one bacteriophage that does not target E. coli under cell-free conditions. The at least one additional bacterial cell lysate may be obtained from a bacterial host species selected from among Bacillus species, Klebsiella species, Pseudomonas species, Acinobacter species, Staphylococcus species, Shigella species, Xanthomonas species, Serratia species, Erwinia species, Ralstonia species, Candidatus Liberibacter species, Mycobacterium species, Escherichia species, Enterococcus species, Enterobacter species, Streptococcus species, Flavobacterium species, Borrelia species, Clostridioides species, Helicobacter species, Salmonella species, Clavibacter species, Campylobacter species, Agrobacterium species, Pectobacterium species, Burkholderia species, Xylella species, Yersinia species, Neisseria species, and Listertia species. In some embodiments, the mixture comprises additional bacterial cell lysates obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 distinct bacterial host species that are not E. coli.

[0008] Additionally or alternatively, in some embodiments of the modified bacterial cell lysate composition disclosed herein, the E. coli lysate comprises about 1% to about 99.9999% of the mixture. In certain embodiments, the E. coli lysate comprises about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 2 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.9%, about 99.99%, about 99.999%, or about 99.9999% of the mixture.

[0009] Additionally or alternatively, in some embodiments of the modified bacterial cell lysate composition disclosed herein, the at least one additional bacterial cell lysate comprises about 0.0001%-99% of the mixture. In certain embodiments, the at least one additional bacterial cell lysate comprises about 0.0001%, about 0.0005%, about 0.001%, about 0.005%, about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% of the mixture.

[0010] In one aspect, the present disclosure provides a modified bacterial cell lysate composition comprising a mixture of an E. coli lysate and genomic DNA derived from a bacterial host species that is not E. coli, wherein the cell lysate composition comprises an effective amount of transcription / translation (TXTL) machinery that is configured to synthesize at least one bacteriophage that does not target E. coli under cell-free conditions. The bacterial host species may be selected from among Bacillus species, Klebsiella species, Pseudomonas species, Acinobacter species, Staphylococcus species, Shigella species, Xanthomonas species, Serratia species, Erwinia species, Ralstonia species, Candidatus Liberibacter species, Mycobacterium species, Escherichia species, Enterococcus species, Enterobacter species, 3 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 Streptococcus species, Flavobacterium species, Borrelia species, Clostridioides species, Helicobacter species, Salmonella species, Clavibacter species, Campylobacter species, Agrobacterium species, Pectobacterium species, Burkholderia species, Xylella species, Yersinia species, Neisseria species, and Listertia species.

[0011] The modified bacterial cell lysate compositions of the present technology may be prepared using one or more of French-press cell lysis, sonication, runoff reactions, or lysate dialysis.

[0012] In another aspect, the present disclosure provides an in vitro method for synthesizing bacteriophage virions comprising contacting a bacteriophage genome with any and all embodiments of the modified bacterial cell lysate compositions disclosed herein, and an energy buffer in vitro to obtain a reaction mixture, and incubating the reaction mixture under conditions to produce viable phage virions, wherein the energy buffer comprises canonical amino acids, phosphoenol pyruvate (PEP), nucleoside triphosphates (NTPs), cofactors, and coenzymes. In some embodiments, the reaction mixture is incubated at 16°C to 42°C for about 1-24 hours. In certain embodiments, the reaction mixture is incubated at 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, or 42°C for about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours. The bacteriophage genome may be isolated from a naturally occurring bacteriophage, or a genetically engineered bacteriophage. In other embodiments, the bacteriophage genome is a synthetic bacteriophage genome.

[0013] In some embodiments, the bacteriophage genome is obtained from a bacteriophage that specifically infects bacterial host cells that are identical to the non- E. coli bacterial host species from which the genomic DNA is derived. In other embodiments, the bacteriophage genome is obtained from a bacteriophage that specifically infects bacterial host cells that are distinct from the non- E. coli bacterial host species from which the genomic DNA is derived. The bacterial host cells may be selected from among Bacillus species, Klebsiella species, Pseudomonas species, Acinobacter species, Staphylococcus species, Shigella species, Xanthomonas species, Serratia species, Erwinia species, Ralstonia species, Candidatus Liberibacter species, Mycobacterium species, Escherichia species, Enterococcus species, Enterobacter species, Streptococcus species, Flavobacterium species, Borrelia species, Clostridioides species, Helicobacter species, Salmonella species, Clavibacter species, Campylobacter species, Agrobacterium species, Pectobacterium species, Burkholderia species, Xylella species, Yersinia 4 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 species, Neisseria species, and Listertia species. Additionally or alternatively, in some embodiments, the bacteriophage genome is obtained from a bacteriophage selected from among T7 phage, T7-like phage, Lambda, K1E, T3, T5, T4, or PhiX174.

[0014] Additionally or alternatively, in some embodiments, the energy buffer further comprises one or more of a polyethylene glycol (PEG) polymer (such as PEG-8000 or PEG- 6000), deoxynucleotide triphosphates (dNTPs), and stabilizers. In certain embodiments, the energy buffer further comprises one or more of Mg-glutamate, K-glutamate, tRNA, cAMP, folinic acid, spermidine, 3-PGA, HEPES, Nicotinamide adenine dinucleotide (NAD), coenzyme A (CoA), DTT, and maltodextrin.

[0015] In any of the preceding embodiments of the methods disclosed herein, the reaction mixture further comprises a vector including a nucleic acid sequence encoding a reporter gene, wherein the nucleic acid sequence encoding the reporter gene is operably linked to a promoter that is responsive to a RNA polymerase (RNAP) encoded by the bacteriophage genome. In certain embodiments, the RNAP is selected from among SP6 RNA Polymerase, T3 RNA Polymerase and T7 RNA polymerase. The reporter gene may encode a bioluminescent protein, a fluorescent protein, or a chemiluminescent protein. Examples of bioluminescent proteins include, but are not limited to, Aequorin, firefly luciferase, Renilla luciferase, red luciferase, luxAB, or nanoluciferase. Examples of chemiluminescent proteins include, but are not limited to, β-galactosidase, horseradish peroxidase (HRP), or alkaline phosphatase. Examples of fluorescent proteins include, but are not limited to, sfGFP, TagBFP, Azurite, EBFP2, mKalamal, Sirius, Sapphire, T-Sapphire, ECFP, Cerulean, SCFP3A, mTurquoise, monomeric Midoriishi- Cyan, TagCFP, mTFP1, EGFP, Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, EYFP, Citrine, Venus, SYFP2, TagYFP, Monomeric Kusabira- Orange, mKOκ, mKO2, mOrange, mOrange2, mRaspberry, mCherry, dsRed, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, mPlum, HcRed-Tandem, mKate2, mNeptune, NirFP, TagRFP657, IFP1.4, iRFP, mKeima Red, LSS-mKate1, LSS- mKate2, PA-GFP, PAmCherryl, PATagRFP, Kaede (green), Kaede (red), KikGR1 (green), KikGR1 (red), PS-CFP2, PS-CFP2, mEos2 (green), mEos2 (red), PSmOrange, or Dronpa.

[0016] In yet another aspect, the present disclosure provides a method for converting a lysogenic bacteriophage into a lytic bacteriophage comprising: generating a plurality of PCR fragments from a DNA template comprising a prophage DNA genome using polymerase chain reaction (PCR), wherein the plurality of PCR fragments collectively span the entire length of the prophage DNA genome except for a gene region encoding integrase; and recombining in vitro the plurality of PCR fragments with a donor heterologous nucleic acid comprising a 5’ flanking 5 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 region and a 3’ flanking region in the presence of a recombination system under conditions to produce a recombinant lytic bacteriophage genome, wherein at least one end of each PCR fragment comprises a sequence that is homologous to (i) an opposite end of another PCR fragment or (ii) the 5’ flanking region of the donor heterologous nucleic acid or (iii) the 3’ flanking region of the donor heterologous nucleic acid. In some embodiments, the donor heterologous nucleic acid replaces the gene region encoding integrase in the recombinant lytic bacteriophage genome.

[0017] Additionally or alternatively, in some embodiments, the methods of the present technology further comprise propagating the recombinant lytic bacteriophage genome in a bacterial host. In other embodiments, the methods of the present technology further comprise contacting the recombinant lytic bacteriophage genome with a bacterial cell lysate, and an energy buffer in vitro to obtain a reaction mixture, and incubating the reaction mixture under conditions to produce viable phage virions, wherein the energy buffer comprises canonical amino acids, phosphoenol pyruvate (PEP), nucleoside triphosphates (NTPs), cofactors, and coenzymes and wherein the cell lysate comprises an effective amount of transcription / translation (TXTL) machinery that is configured to synthesize bacteriophage under cell-free conditions.

[0018] In one aspect, the present disclosure provides a method for generating a genetically modified bacteriophage species comprising generating a plurality of PCR fragments from a DNA template comprising a bacteriophage DNA genome using polymerase chain reaction (PCR), wherein the plurality of PCR fragments collectively span the entire length of the bacteriophage DNA genome except for a first gene region; and recombining in vitro the plurality of PCR fragments with a donor heterologous nucleic acid comprising a 5’ flanking region and a 3’ flanking region in the presence of a recombination system under conditions to produce a genetically modified bacteriophage genome, wherein at least one end of each PCR fragment comprises a sequence that is homologous to (i) an opposite end of another PCR fragment or (ii) the 5’ flanking region of the donor heterologous nucleic acid or (iii) the 3’ flanking region of the donor heterologous nucleic acid, and wherein the donor heterologous nucleic acid replaces the first gene region in the genetically modified bacteriophage genome. In any and all embodiments of the methods disclosed herein, the genetically modified bacteriophage species is genetically modified T7 phage. In some embodiments, the genetically modified bacteriophage species lacks endotoxin genes (e.g., LpxL1, LpxL2).

[0019] In certain embodiments, the method further comprises propagating the genetically modified bacteriophage genome in a bacterial host. In other embodiments, the method further comprises contacting the genetically modified bacteriophage genome with a bacterial cell lysate, 6 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 and an energy buffer in vitro to obtain a reaction mixture, and incubating the reaction mixture under conditions to produce viable phage virions, wherein the energy buffer comprises canonical amino acids, phosphoenol pyruvate (PEP), nucleoside triphosphates (NTPs), cofactors, and coenzymes and wherein the cell lysate comprises an effective amount of transcription / translation (TXTL) machinery that is configured to synthesize bacteriophage under cell-free conditions. The bacterial cell lysate is obtained from naturally occurring bacterial host cell or a genetically modified bacterial host cell. In certain embodiments, the genetically modified bacterial host cell overexpresses one or more of translation initiation factor IF-3 (infC), OxyS and CyaR and / or represses RecC subunit exonuclease RecBCD. In other embodiments, the bacterial cell lysate comprises any and all embodiments of the modified bacterial cell lysate compositions disclosed herein.

[0020] Additionally or alternatively, in some embodiments, the energy buffer further comprises one or more of a polyethylene glycol (PEG) polymer (such as PEG-8000 or PEG- 6000), deoxynucleotide triphosphates (dNTPs), and stabilizers. In certain embodiments, the energy buffer further comprises one or more of Mg-glutamate, K-glutamate, tRNA, cAMP, folinic acid, spermidine, 3-PGA, HEPES, Nicotinamide adenine dinucleotide (NAD), coenzyme A (CoA), DTT, and maltodextrin.

[0021] In any of the preceding embodiments of the methods disclosed herein, the reaction mixture further comprises a vector including a nucleic acid sequence encoding a reporter gene, wherein the nucleic acid sequence encoding the reporter gene is operably linked to a promoter that is responsive to a RNA polymerase (RNAP) encoded by the bacteriophage genome. In certain embodiments, the RNAP is selected from among SP6 RNA Polymerase, T3 RNA Polymerase and T7 RNA polymerase. The reporter gene may encode a bioluminescent protein, a fluorescent protein, or a chemiluminescent protein. Examples of bioluminescent proteins include, but are not limited to, Aequorin, firefly luciferase, Renilla luciferase, red luciferase, luxAB, or nanoluciferase. Examples of chemiluminescent proteins include, but are not limited to, β-galactosidase, horseradish peroxidase (HRP), or alkaline phosphatase. Examples of fluorescent proteins include, but are not limited to, sfGFP, TagBFP, Azurite, EBFP2, mKalamal, Sirius, Sapphire, T-Sapphire, ECFP, Cerulean, SCFP3A, mTurquoise, monomeric Midoriishi- Cyan, TagCFP, mTFP1, EGFP, Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, EYFP, Citrine, Venus, SYFP2, TagYFP, Monomeric Kusabira- Orange, mKOκ, mKO2, mOrange, mOrange2, mRaspberry, mCherry, dsRed, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, mPlum, HcRed-Tandem, mKate2, mNeptune, NirFP, TagRFP657, IFP1.4, iRFP, mKeima Red, LSS-mKate1, LSS- 7 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 mKate2, PA-GFP, PAmCherryl, PATagRFP, Kaede (green), Kaede (red), KikGR1 (green), KikGR1 (red), PS-CFP2, PS-CFP2, mEos2 (green), mEos2 (red), PSmOrange, or Dronpa.

[0022] In any and all embodiments of the methods disclosed herein, the recombination system comprises a 5’-3’ exonuclease, a DNA polymerase, and a DNA ligase. In any of the preceding embodiments of the methods disclosed herein, the donor heterologous nucleic acid comprises an open reading frame that encodes a bioluminescent protein, a fluorescent protein, a chemiluminescent protein, a heterologous bactericidal gene, a phage protein that modifies host range, or any combination thereof. In certain embodiments, the open reading frame of the donor heterologous nucleic acid is operably linked to an expression control sequence that is capable of directing expression of the bioluminescent protein, the fluorescent protein, the chemiluminescent protein, the heterologous bactericidal gene, a phage protein that modifies host range, or any combination thereof. The expression control sequence may be an inducible promoter or a constitutive promoter. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIG.1: Schematic of Cell-free bacteriophage synthesis

[0024] FIGs.2A-2J: Approaches to diversified cell-free bacteriophage synthesis. FIG. 2A: Rebooting phage with standardized E. coli-TXTL from phage genomes (gDNA); FIG.2B: Establish a standard Gram (-) TXTL (e.g. E. coli BL21) and equivalent high-productivity Gram (+) TXTL (e.g. B. megaterium) for rebooting corresponding Gram (-) and (+) phage respectively; FIG.2C: Standardized E. coli-TXTL supplemented with pre-identified essential host protein (co-expressed or exogenous); FIG.2D: Standardized E. coli-TXTL supplemented with non-E. coli cell-lysate to provide non-preidentified host factors to reboot non-coliphage; FIG.2E: representation of diverse bacteria specifically targeted by diverse phage; FIG.2F: Establish a Genus-level CFBS ; FIG.2G: “Universal” CFBS comprised of a mixture of diverse cell-lysates; FIG.2H: in vitro phage self-assembly by mixing phage gDNA with pre-expressed capsid protein in buffer + ATP; FIG.2I: CFBS by decoupled transcription (TX) and translation (TL) by generating phage mRNAs through in vitro transcription to then be added to standardized cell- extracts for translation and phage assembly; FIG.2J: Establish a cell-free mimic proteome to phage host to provide essential host factors by (1) cell-free expression of the host proteome directed by its whole genome then (2) rebooting phage from its gDNA. Of these approaches, FIG.2A, FIG.2C, FIG.2H, and FIG.2I have been demonstrated as feasible in peer reviewed research. See Mark Rustad et al., Synthetic Biology, Volume 3, Issue 1, 2018, ysy002, https: / / doi.org / 10.1093 / synbio / ysy002; Cell-free production of personalized therapeutic phages 8 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 targeting multidrug-resistant bacteria. https: / / doi.org / 10.1016 / j.chembiol.2022.06.003; Lauren R.H. Krumpe, et al. (2006) Proteomics 6, 4210-4222; Weise LI et al., Nucleic Acids Res.2019 Nov 18;47(20):10956-10967. TXTL of non-endogenous genomes in artificial cell environments has been described in Deyama et al., Chem. Commun., 2021, 57, 10367; Fujiwara et al., Nucleic Acids Research, 2017, Vol.45, No.1911449–11458.

[0025] FIGs.3A-3B: Conversion of lysogenic to lytic phage by genome engineering. FIG. 3A: Synthetic phage genomes assembled in vitro. Prophage sequences are identified in lysogens and lysogeny genes (e.g. integrase) are annotated. Synthetic genomes are assembled from genome fragments synthesized omitting lysogeny genes including any desired heterologous genes (in pink). FIG.3B: Engineered lytic phage rebooted from synthetic genomes by (4a) transformation into competent host / stepping-stone bacteria or (4b) in vitro by cell-free bacteriophage synthesis.

[0026] FIGs.4A-4B: Rebooting phage from synthetic genomes. FIG.4A: Phage genome fragments are PCR amplified with flanking DNA homologous to adjacent genomic regions (here fragments A, B, C, D). Synthetic genomes are combined by Gibson assembly. Heterologous genes may be incorporated by generating PCR fragments with appropriate overhangs. Here, fragment C is replaced by fragments E and F (blue) and heterologous gene (yellow) inserted between. FIG.4B: Synthetic genomes are rebooted into active phage in vivo by transformation into competent host bacteria (top) or in vitro using cell-free bacteriophage synthesis (bottom). Created with Biorender.com.

[0027] FIGs.5A-5C: Rapid rebooting of T7_ClyF and T7_sfGFP from synthetic genomes. FIG.5A: Progress of Gibson assembly of synthetic T7 genomes (from PCR fragments A, B, E, expression cassette, F, and D) from 0-60 min. FIG.5B: Quick confirmation of successful CFBS rebooting of synthetic T7 by detecting sfGFP expression after infection of BL21 with rebooted phage. Fluorescence was measured as an endpoint after 30 min infection of BL21 with phage taken directly from CFBS reactions. LB medium was used as a negative control. FIG.5C: Spot tests of antimicrobial activity of synthetic T7 against MRSA, E. coli BL21, and co-culture overlay plates. Purified T7 stocks (T7_WT) were included as a control. RFU Differences between LB and synthetic phage cultures were evaluated by unpaired t-test (significance threshold p<0.05, *; p<0.0001, ****). Error bars reflect standard deviations of n=3 replicates. T7_ClyF thus has activity against E. coli and MRSA.

[0028] FIG.6: Recent non-model bacteria TXTL cell-free expression system setup, processing, and protein yields. 9 4859-9106-8900.1Atty. Dkt. No.: 136669-0119

[0029] FIG.7: Recent non-model bacteria TXTL cell-free expression system setup, processing, and protein yields.

[0030] FIG.8: Cell-free Bacteriophage Synthesis: Phage Characteristics, Yields, and Efficiency. DETAILED DESCRIPTION

[0031] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology.

[0032] In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, microbiology and recombinant DNA are used. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel et al. eds. (2007) Current Protocols in Molecular Biology; the series Methods in Enzymology (Academic Press, Inc., N.Y.); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane eds. (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No.4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds (1996) Weir’s Handbook of Experimental Immunology.

[0033] The modified bacterial cell lysate compositions of the present technology confers the following advantages over conventional phage engineering techniques: (1) the modified bacterial cell lysate compositions enable the study pathogenic or fastidious phage transcription / translation and promotes high-yield protein expression; (2) the modified bacterial cell lysate compositions can be used to screen for and optimize gene expression using endogenous transcription regulatory elements; and (3) the modified bacterial cell lysate compositions promotes expression of endogenous gene clusters for chemical synthesis without the need for extensive genetic engineering. 10 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 Definitions

[0034] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a cell” includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art.

[0035] As used herein, the term “about” in reference to a number is generally taken to include numbers that fall within a range of 1%, 5%, or 10% in either direction (greater than or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).

[0036] The term “amino acid” refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally encoded amino acids are the 20 canonical amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrolysine and selenocysteine. Amino acid analogs refer to agents that have the same basic chemical structure as a naturally occurring amino acid, i.e., an α carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, such as, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (such as, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. In some embodiments, amino acids forming a polypeptide are in the D form. In some embodiments, the amino acids forming a polypeptide are in the L form. In some embodiments, a first plurality of amino acids forming a polypeptide is in the D form and a second plurality is in the L form.

[0037] Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, are referred to by their commonly accepted single-letter code.

[0038] As used herein, “bacteriophage” or “phage” refers to a virus that infects bacteria. Bacteriophages are obligate intracellular parasites that multiply inside bacteria by co-opting 11 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 some or all of the host biosynthetic machinery (i.e., viruses that infect bacteria). Though different bacteriophages may contain different materials, they all contain nucleic acid and protein, and can under certain circumstances be encapsulated in a lipid membrane. Depending upon the phage, the nucleic acid can be either DNA or RNA (but not both) and can exist in various forms.

[0039] As used herein, the term “effective amount” refers to a quantity sufficient to achieve a desired effect or outcome, e.g., an amount which results in the manufacturing or synthesis of bacteriophage under in vitro cell-free conditions, e.g., cell-free bacteriophage synthesis (CFBS).

[0040] As used herein, “expression” includes one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and / or other modifications of the translation product, if required for proper expression and function.

[0041] As used herein, an “expression control sequence” refers to polynucleotide sequences which are necessary to affect the expression of coding sequences to which they are operably linked. Expression control sequences are sequences which control the transcription, post- transcriptional events and translation of nucleic acid sequences. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence. The term “control sequences” is intended to encompass, at a minimum, any component whose presence is essential for expression, and can also encompass an additional component whose presence is advantageous, for example, leader sequences.

[0042] As used herein, “heterologous nucleic acid sequence” is any sequence placed at a location in the genome where it does not normally occur. A heterologous nucleic acid sequence may comprise a sequence that does not naturally occur in a bacteriophage, or it may comprise only sequences naturally found in the bacteriophage, but placed at a non-normally occurring location in the genome. In some embodiments, the heterologous nucleic acid sequence is not a natural phage sequence. In certain embodiments, the heterologous nucleic acid sequence is a 12 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 natural phage sequence that is derived from a different phage. In other embodiments, the heterologous nucleic acid sequence is a sequence that occurs naturally in the genome of a wild- type phage but is then relocated to another site where it does not naturally occur, rendering it a heterologous sequence at that new site.

[0043] “Homology” or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same nucleobase or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) has a certain percentage (for example, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of “sequence identity” to another sequence means that, when aligned, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art. In some embodiments, default parameters are used for alignment. One alignment program is BLAST, using default parameters. In particular, programs are BLASTN and BLASTP, using the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by ═HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR. Details of these programs can be found at the National Center for Biotechnology Information. Biologically equivalent polynucleotides are those having the specified percent homology and encoding a polypeptide having the same or similar biological activity. Two sequences are deemed “unrelated” or “non-homologous” if they share less than 40% identity, or less than 25% identity, with each other.

[0044] As used herein, a “host cell” is a bacterial cell that can be infected by a phage to yield progeny phage particles. A host cell can form phage particles from a particular type of phage genomic DNA. In some embodiments, the phage genomic DNA is introduced into the host cell by infecting the host cell with a phage. In some embodiments, the phage genomic DNA is introduced into the host cell using transformation, electroporation, or any other suitable technique. In some embodiments, the phage genomic DNA is substantially pure when introduced into the host cell. In some embodiments, the phage genomic DNA is present in a vector when introduced into the host cell. The definition of host cell can vary from one phage to 13 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 another. For example, E. coli may be the natural host cell for a particular type of phage, but Klebsiella pneumoniae is not.

[0045] As used herein, the term “isolated” refers to a substance or entity that has been separated from at least some of the components with which it was associated when initially produced (whether in nature or in an experimental setting). Isolated substances and / or entities may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were initially associated. In some embodiments, isolated substances and / or entities are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is “pure” if it is substantially free of other components.

[0046] As used herein, “operably linked” means that expression control sequences are positioned relative to the nucleic acid of interest to initiate, regulate or otherwise control transcription of the nucleic acid of interest.

[0047] As used herein, a “phage genome” includes naturally occurring phage genomes and derivatives thereof. Generally, the derivatives possess the ability to propagate in the same hosts as the naturally occurring phage. In some embodiments, the only difference between a naturally occurring phage genome and a derivative phage genome is at least one of a deletion and an addition of nucleotides from at least one end of the phage genome (if the genome is linear) or at least one point in the genome (if the genome is circular).

[0048] As used herein, the term “polynucleotide” or “nucleic acid” means any RNA or DNA, which may be unmodified or modified RNA or DNA. Polynucleotides include, without limitation, single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, RNA that is mixture of single- and double- stranded regions, and hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.

[0049] As used herein, the term “recombinant” when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the material is derived from a cell so modified. Thus, for 14 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.

[0050] As used herein, an endogenous nucleic acid sequence in the genome of an organism (or the encoded protein product of that sequence) is deemed “recombinant” herein if a heterologous sequence is placed adjacent to the endogenous nucleic acid sequence, such that the expression of this endogenous nucleic acid sequence is altered. In this context, a heterologous sequence is a sequence that is not naturally adjacent to the endogenous nucleic acid sequence, whether or not the heterologous sequence is itself endogenous to the organism (originating from the same organism or progeny thereof) or exogenous (originating from a different organism or progeny thereof). By way of example, a promoter sequence can be substituted (e.g., by homologous recombination) for the native promoter of a gene in the genome of an organism, such that this gene has an altered expression pattern. This gene would be “recombinant” because it is separated from at least some of the sequences that naturally flank it. A nucleic acid is also considered “recombinant” if it contains any modifications that do not naturally occur in the corresponding nucleic acid in a genome. For instance, an endogenous coding sequence is considered “recombinant” if it contains an insertion, deletion or a point mutation introduced artificially, e.g., by human intervention. A “recombinant nucleic acid” also includes a nucleic acid integrated into a host cell chromosome at a heterologous site and a nucleic acid construct present as an episome.

[0051] As used herein, a “recombinant bacteriophage genome” is a bacteriophage genome that has been genetically modified by the insertion of a heterologous nucleic acid sequence into the bacteriophage genome. A “recombinant bacteriophage” means a bacteriophage that comprises a recombinant bacteriophage genome. In some embodiments, the bacteriophage genome is modified by recombinant DNA technology to introduce a heterologous nucleic acid sequence into the genome at a defined site. In some embodiments, the heterologous nucleic acid sequence is introduced with no corresponding loss of endogenous phage genomic nucleotides. In other words, if bases N1 and N2 are adjacent in the wild-type bacteriophage genome, the heterologous nucleic acid sequence is inserted between N1 and N2. Thus, in the resulting recombinant bacteriophage genome, the heterologous nucleic acid sequence is flanked by nucleotides N1 and N2. In some embodiments, endogenous phage nucleotides are removed or replaced during the insertion of the heterologous nucleic acid sequence. For example, in some embodiments, the heterologous nucleic acid sequence is inserted in place of some or all of the endogenous phage sequence which is removed. In some embodiments, endogenous phage 15 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 sequences are removed from a position in the phage genome distant from the site(s) of insertion of the heterologous nucleic acid sequences.

[0052] As used herein, the term “sample” refers to clinical samples obtained from a subject or isolated microorganisms. In certain embodiments, a sample is obtained from a biological source (i.e., a "biological sample"), such as tissue, bodily fluid, or microorganisms collected from a subject. Sample sources include, but are not limited to, mucus, sputum, bronchial alveolar lavage (BAL), bronchial wash (BW), whole blood, bodily fluids, cerebrospinal fluid (CSF), urine, plasma, serum, or tissue. Bacteriophage

[0053] Bacteriophage are obligate intracellular parasites that multiply inside bacteria by co- opting some or all of the host biosynthetic machinery. Phages contain nucleic acid and protein, and may be enveloped by a lipid membrane. Depending upon the phage, the nucleic acid genome can be either DNA or RNA but not both, and can exist in either circular or linear forms. The size of the phage genome varies depending upon the phage. The simplest phages have genomes that are only a few thousand nucleotides in size, while the more complex phages may contain more than 100,000 nucleotides in their genome, and in rare instances no more than 500,000 bp. The number and amount of individual types of protein in phage particles will vary depending upon the phage. The proteins function in infection and to protect the nucleic acid genome from environmental nucleases.

[0054] Phage genomes come in a variety of sizes and shapes (e.g., linear or circular). Most phages range in size from 24-200 nm in diameter. The capsid is composed of many copies of one or more phage proteins, and acts as a protective envelope around the phage genome. Many phages have tails attached to the phage capsid. The tail is a hollow tube through which the phage nucleic acid passes during infection. The size of the tail can vary and some phages do not even have a tail structure. In the more complex phages, the tail is surrounded by a contractile sheath which contracts during infection of the bacterial host cell. At the end of the tail, phages have a base plate and one or more tail fibers attached to it. The base plate and tail fibers are involved in the binding of the phage to the host cell.

[0055] Lytic or virulent phages are phages which can only multiply in bacteria and lyse the bacterial host cell at the end of the life cycle of the phage. The lifecycle of a lytic phage begins with an eclipse period. During the eclipse phase, no infectious phage particles can be found either inside or outside the host cell. The phage nucleic acid takes over the host biosynthetic machinery and phage specific mRNAs and proteins are produced. Early phage mRNAs code for 16 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 early proteins that are needed for phage DNA synthesis and for shutting off host DNA, RNA and protein biosynthesis. In some cases, the early proteins actually degrade the host chromosome. After phage DNA is made late mRNAs and late proteins are made. The late proteins are the structural proteins that comprise the phage as well as the proteins needed for lysis of the bacterial cell. In the next phase, the phage nucleic acid and structural proteins are assembled and infectious phage particles accumulate within the cell. The bacteria begin to lyse due to the accumulation of the phage lysis protein, leading to the release of intracellular phage particles. The number of particles released per infected cell can be as high as 1000 or more. Lytic phage may be enumerated by a plaque assay. The assay is performed at a low enough concentration of phage such that each plaque arises from a single infectious phage. The infectious particle that gives rise to a plaque is called a PFU (plaque forming unit).

[0056] Lysogenic phages are those that can either multiply via the lytic cycle or enter a quiescent state in the host cell. In the quiescent state, the phage genome exists as a prophage (i.e., it has the potential to produce phage). In most cases, the phage DNA actually integrates into the host chromosome and is replicated along with the host chromosome and passed on to the daughter cells. The host cell harboring a prophage is not adversely affected by the presence of the prophage and the lysogenic state may persist indefinitely. The lysogenic state can be terminated upon exposure to adverse conditions. Conditions which favor the termination of the lysogenic state include: desiccation, exposure to UV or ionizing radiation, exposure to mutagenic chemicals, etc. Adverse conditions lead to the production of proteases (rec A protein), the expression of the phage genes, reversal of the integration process, and lytic multiplication.

[0057] In some embodiments, a phage genome comprises at least 5 kilobases (kb), at least 10 kb, at least 15 kb, at least 20 kb, at least 25 kb, at least 30 kb, at least 35 kb, at least 40 kb, at least 45 kb, at least 50 kb, at least 55 kb, at least 60 kb, at least 65 kb, at least 70 kb, at least 75 kb, at least 80 kb, at least 85 kb, at least 90 kb, at least 95 kb, at least 100 kb, at least 105 kb, at least 110 kb, at least 115 kb, at least 120 kb, at least 125 kb, at least 130 kb, at least 135 kb, at least 140 kb, at least 145 kb, at least 150 kb, at least 175 kb, at least 200 kb, at least 225 kb, at least 250 kb, at least 275 kb, at least 300 kb, at least 325 kb, at least 350 kb, at least 375 kb, at least 400 kb, at least 425 kb, at least 450 kb, at least 475 kb, or at least 500 kb of nucleic acids. Cell-free Bacteriophage Synthesis Compositions and Methods of the Present Technology

[0058] In one aspect, the present disclosure provides a modified bacterial cell lysate composition comprising a mixture of an E. coli lysate and at least one additional bacterial cell lysate obtained from a bacterial host species that is not E. coli, wherein the cell lysate composition comprises an effective amount of transcription / translation (TXTL) machinery that is 17 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 configured to synthesize at least one bacteriophage that does not target E. coli under cell-free conditions. The at least one additional bacterial cell lysate may be obtained from a bacterial host species selected from among Bacillus species, Klebsiella species, Pseudomonas species, Acinobacter species, Staphylococcus species, Shigella species, Xanthomonas species, Serratia species, Erwinia species, Ralstonia species, Candidatus Liberibacter species, Mycobacterium species, Escherichia species, Enterococcus species, Enterobacter species, Streptococcus species, Flavobacterium species, Borrelia species, Clostridioides species, Helicobacter species, Salmonella species, Clavibacter species, Campylobacter species, Agrobacterium species, Pectobacterium species, Burkholderia species, Xylella species, Yersinia species, Neisseria species, and Listertia species. In some embodiments, the mixture comprises additional bacterial cell lysates obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 distinct bacterial host species that are not E. coli.

[0059] Additionally or alternatively, in some embodiments of the modified bacterial cell lysate composition disclosed herein, the E. coli lysate comprises about 1% to about 99.9999% of the mixture. In certain embodiments, the E. coli lysate comprises about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.9%, about 99.99%, about 99.999%, or about 99.9999% of the mixture.

[0060] Additionally or alternatively, in some embodiments of the modified bacterial cell lysate composition disclosed herein, the at least one additional bacterial cell lysate comprises about 0.0001%-99% of the mixture. In certain embodiments, the at least one additional bacterial cell lysate comprises about 0.0001%, about 0.0005%, about 0.001%, about 0.005%, about 0.01%, about 0.05%, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, 18 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, about 46%, about 47%, about 48%, about 49%, about 50%, about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% of the mixture.

[0061] In one aspect, the present disclosure provides a modified bacterial cell lysate composition comprising a mixture of an E. coli lysate and genomic DNA derived from a bacterial host species that is not E. coli, wherein the cell lysate composition comprises an effective amount of transcription / translation (TXTL) machinery that is configured to synthesize at least one bacteriophage that does not target E. coli under cell-free conditions. The bacterial host species may be selected from among Bacillus species, Klebsiella species, Pseudomonas species, Acinobacter species, Staphylococcus species, Shigella species, Xanthomonas species, Serratia species, Erwinia species, Ralstonia species, Candidatus Liberibacter species, Mycobacterium species, Escherichia species, Enterococcus species, Enterobacter species, Streptococcus species, Flavobacterium species, Borrelia species, Clostridioides species, Helicobacter species, Salmonella species, Clavibacter species, Campylobacter species, Agrobacterium species, Pectobacterium species, Burkholderia species, Xylella species, Yersinia species, Neisseria species, and Listertia species.

[0062] The modified bacterial cell lysate compositions of the present technology may be prepared using one or more of French-press cell lysis, sonication, runoff reactions, or lysate dialysis.

[0063] In another aspect, the present disclosure provides an in vitro method for synthesizing bacteriophage virions comprising contacting a bacteriophage genome with any and all embodiments of the modified bacterial cell lysate compositions disclosed herein, and an energy buffer in vitro to obtain a reaction mixture, and incubating the reaction mixture under conditions to produce viable phage virions, wherein the energy buffer comprises canonical amino acids, phosphoenol pyruvate (PEP), nucleoside triphosphates (NTPs), cofactors, and coenzymes. In 19 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 some embodiments, the reaction mixture is incubated at 16°C to 42°C for about 1-24 hours. In certain embodiments, the reaction mixture is incubated at 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, or 42°C for about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, or 24 hours. The bacteriophage genome may be isolated from a naturally occurring bacteriophage, or a genetically engineered bacteriophage. In other embodiments, the bacteriophage genome is a synthetic bacteriophage genome.

[0064] In some embodiments, the bacteriophage genome is obtained from a bacteriophage that specifically infects bacterial host cells that are identical to the non- E. coli bacterial host species from which the genomic DNA is derived. In other embodiments, the bacteriophage genome is obtained from a bacteriophage that specifically infects bacterial host cells that are distinct from the non- E. coli bacterial host species from which the genomic DNA is derived. The bacterial host cells may be selected from among Bacillus species, Klebsiella species, Pseudomonas species, Acinobacter species, Staphylococcus species, Shigella species, Xanthomonas species, Serratia species, Erwinia species, Ralstonia species, Candidatus Liberibacter species, Mycobacterium species, Escherichia species, Enterococcus species, Enterobacter species, Streptococcus species, Flavobacterium species, Borrelia species, Clostridioides species, Helicobacter species, Salmonella species, Clavibacter species, Campylobacter species, Agrobacterium species, Pectobacterium species, Burkholderia species, Xylella species, Yersinia species, Neisseria species, and Listertia species. Additionally or alternatively, in some embodiments, the bacteriophage genome is obtained from a bacteriophage selected from among T7 phage, T7-like phage, Lambda, K1E, T3, T5, T4, or PhiX174.

[0065] Additionally or alternatively, in some embodiments, the energy buffer further comprises one or more of a polyethylene glycol (PEG) polymer (such as PEG-8000 or PEG- 6000), deoxynucleotide triphosphates (dNTPs), and stabilizers. In certain embodiments, the energy buffer further comprises one or more of Mg-glutamate, K-glutamate, tRNA, cAMP, folinic acid, spermidine, 3-PGA, HEPES, Nicotinamide adenine dinucleotide (NAD), coenzyme A (CoA), DTT, and maltodextrin.

[0066] In any of the preceding embodiments of the methods disclosed herein, the reaction mixture further comprises a vector including a nucleic acid sequence encoding a reporter gene, wherein the nucleic acid sequence encoding the reporter gene is operably linked to a promoter that is responsive to a RNA polymerase (RNAP) encoded by the bacteriophage genome. In 20 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 certain embodiments, the RNAP is selected from among SP6 RNA Polymerase, T3 RNA Polymerase and T7 RNA polymerase. The reporter gene may encode a bioluminescent protein, a fluorescent protein, or a chemiluminescent protein. Examples of bioluminescent proteins include, but are not limited to, Aequorin, firefly luciferase, Renilla luciferase, red luciferase, luxAB, or nanoluciferase. Examples of chemiluminescent proteins include, but are not limited to, β-galactosidase, horseradish peroxidase (HRP), or alkaline phosphatase. Examples of fluorescent proteins include, but are not limited to, sfGFP, TagBFP, Azurite, EBFP2, mKalamal, Sirius, Sapphire, T-Sapphire, ECFP, Cerulean, SCFP3A, mTurquoise, monomeric Midoriishi- Cyan, TagCFP, mTFP1, EGFP, Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, EYFP, Citrine, Venus, SYFP2, TagYFP, Monomeric Kusabira- Orange, mKOκ, mKO2, mOrange, mOrange2, mRaspberry, mCherry, dsRed, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, mPlum, HcRed-Tandem, mKate2, mNeptune, NirFP, TagRFP657, IFP1.4, iRFP, mKeima Red, LSS-mKate1, LSS- mKate2, PA-GFP, PAmCherryl, PATagRFP, Kaede (green), Kaede (red), KikGR1 (green), KikGR1 (red), PS-CFP2, PS-CFP2, mEos2 (green), mEos2 (red), PSmOrange, or Dronpa.

[0067] In yet another aspect, the present disclosure provides a method for converting a lysogenic bacteriophage into a lytic bacteriophage comprising: generating a plurality of PCR fragments from a DNA template comprising a prophage DNA genome using polymerase chain reaction (PCR), wherein the plurality of PCR fragments collectively span the entire length of the prophage DNA genome except for a gene region encoding integrase; and recombining in vitro the plurality of PCR fragments with a donor heterologous nucleic acid comprising a 5’ flanking region and a 3’ flanking region in the presence of a recombination system under conditions to produce a recombinant lytic bacteriophage genome, wherein at least one end of each PCR fragment comprises a sequence that is homologous to (i) an opposite end of another PCR fragment or (ii) the 5’ flanking region of the donor heterologous nucleic acid or (iii) the 3’ flanking region of the donor heterologous nucleic acid. In some embodiments, the donor heterologous nucleic acid replaces the gene region encoding integrase in the recombinant lytic bacteriophage genome.

[0068] Additionally or alternatively, in some embodiments, the methods of the present technology further comprise propagating the recombinant lytic bacteriophage genome in a bacterial host. In other embodiments, the methods of the present technology further comprise contacting the recombinant lytic bacteriophage genome with a bacterial cell lysate, and an energy buffer in vitro to obtain a reaction mixture, and incubating the reaction mixture under conditions to produce viable phage virions, wherein the energy buffer comprises canonical 21 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 amino acids, phosphoenol pyruvate (PEP), nucleoside triphosphates (NTPs), cofactors, and coenzymes and wherein the cell lysate comprises an effective amount of transcription / translation (TXTL) machinery that is configured to synthesize bacteriophage under cell-free conditions.

[0069] In one aspect, the present disclosure provides a method for generating a genetically modified bacteriophage species comprising generating a plurality of PCR fragments from a DNA template comprising a bacteriophage DNA genome using polymerase chain reaction (PCR), wherein the plurality of PCR fragments collectively span the entire length of the bacteriophage DNA genome except for a first gene region; and recombining in vitro the plurality of PCR fragments with a donor heterologous nucleic acid comprising a 5’ flanking region and a 3’ flanking region in the presence of a recombination system under conditions to produce a genetically modified bacteriophage genome, wherein at least one end of each PCR fragment comprises a sequence that is homologous to (i) an opposite end of another PCR fragment or (ii) the 5’ flanking region of the donor heterologous nucleic acid or (iii) the 3’ flanking region of the donor heterologous nucleic acid, and wherein the donor heterologous nucleic acid replaces the first gene region in the genetically modified bacteriophage genome. In any and all embodiments of the methods disclosed herein, the genetically modified bacteriophage species is genetically modified T7 phage. In some embodiments, the genetically modified bacteriophage species lacks endotoxin genes (e.g., LpxL1, LpxL2).

[0070] In any of the above embodiments of the methods disclosed herein, the homologous 5’ flanking region of the heterologous nucleic acid has a length of about 20-30 base pairs (bps), 30- 40 bps, 40-50 bps, 50-60 bps, 60-70 bps, 70-80 bps, 80-90 bps, 90-100 bps, 100-110 bps, 110- 120 bps, 120-130 bps, 130-140 bps, 140-150 bps, 150-160 bps, 160-170 bps, 170-180 bps, 180- 190 bps, 190-200 bps, 200-210 bps, 210-220 bps, 220-230 bps, 230-240 bps, 240-250 bps, 250- 260 bps, 260-270 bps, 270-280 bps, 280-290 bps, 290-300 bps, 300-310 bps, 310- 320 bps, 320- 330 bps, 330-340 bps, 340-350 bps, 350-360 bps, 360-370 bps, 370-380 bps, 380-390 bps, 390- 400 bps, 400-410 bps, 410-420 bps, 420-430 bps, 430-440 bps, 440-450 bps, 450-460 bps, 460- 470 bps, 470-480 bps, 480-490 bps, 490-500 bps, 500-510 bps, 510-520 bps, 520-530 bps, 530- 540 bps, 540-550 bps, 550-560 bps, 560-570 bps, 570-580 bps, 580-590 bps, or 590-600 bps.

[0071] Additionally or alternatively, in any of the above embodiments of the methods disclosed herein, the homologous 3’ flanking region of the heterologous nucleic acid has a length of about 20-30 base pairs (bps), 30-40 bps, 40-50 bps, 50-60 bps, 60-70 bps, 70-80 bps, 80-90 bps, 90-100 bps, 100-110 bps, 110-120 bps, 120-130 bps, 130-140 bps, 140-150 bps, 150-160 bps, 160-170 bps, 170-180 bps, 180-190 bps, 190-200 bps, 200-210 bps, 210-220 bps, 220-230 bps, 230-240 bps, 240-250 bps, 250-260 bps, 260-270 bps, 270-280 bps, 280-290 bps, 290-300 22 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 bps, 300-310 bps, 310-320 bps, 320-330 bps, 330-340 bps, 340-350 bps, 350-360 bps, 360-370 bps, 370-380 bps, 380-390 bps, 390-400 bps, 400-410 bps, 410-420 bps, 420-430 bps, 430-440 bps, 440-450 bps, 450-460 bps, 460-470 bps, 470-480 bps, 480-490 bps, 490-500 bps, 500-510 bps, 510-520 bps, 520-530 bps, 530-540 bps, 540-550 bps, 550-560 bps, 560-570 bps, 570-580 bps, 580-590 bps, or 590-600 bps.

[0072] In certain embodiments, the method further comprises propagating the genetically modified bacteriophage genome in a bacterial host. In other embodiments, the method further comprises contacting the genetically modified bacteriophage genome with a bacterial cell lysate, and an energy buffer in vitro to obtain a reaction mixture, and incubating the reaction mixture under conditions to produce viable phage virions, wherein the energy buffer comprises canonical amino acids, phosphoenol pyruvate (PEP), nucleoside triphosphates (NTPs), cofactors, and coenzymes and wherein the cell lysate comprises an effective amount of transcription / translation (TXTL) machinery that is configured to synthesize bacteriophage under cell-free conditions. The bacterial cell lysate is obtained from naturally occurring bacterial host cell or a genetically modified bacterial host cell. In certain embodiments, the genetically modified bacterial host cell overexpresses one or more of translation initiation factor IF-3 (infC), OxyS and CyaR and / or represses RecC subunit exonuclease RecBCD. In other embodiments, the bacterial cell lysate comprises any and all embodiments of the modified bacterial cell lysate compositions disclosed herein.

[0073] Additionally or alternatively, in some embodiments, the energy buffer further comprises one or more of a polyethylene glycol (PEG) polymer (such as PEG-8000 or PEG- 6000), deoxynucleotide triphosphates (dNTPs), and stabilizers. In certain embodiments, the energy buffer further comprises one or more of Mg-glutamate, K-glutamate, tRNA, cAMP, folinic acid, spermidine, 3-PGA, HEPES, Nicotinamide adenine dinucleotide (NAD), coenzyme A (CoA), DTT, and maltodextrin.

[0074] In any of the preceding embodiments of the methods disclosed herein, the reaction mixture further comprises a vector including a nucleic acid sequence encoding a reporter gene, wherein the nucleic acid sequence encoding the reporter gene is operably linked to a promoter that is responsive to a RNA polymerase (RNAP) encoded by the bacteriophage genome. In certain embodiments, the RNAP is selected from among SP6 RNA Polymerase, T3 RNA Polymerase and T7 RNA polymerase. The reporter gene may encode a bioluminescent protein, a fluorescent protein, or a chemiluminescent protein. Examples of bioluminescent proteins include, but are not limited to, Aequorin, firefly luciferase, Renilla luciferase, red luciferase, luxAB, or nanoluciferase. Examples of chemiluminescent proteins include, but are not limited 23 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 to, β-galactosidase, horseradish peroxidase (HRP), or alkaline phosphatase. Examples of fluorescent proteins include, but are not limited to, sfGFP, TagBFP, Azurite, EBFP2, mKalamal, Sirius, Sapphire, T-Sapphire, ECFP, Cerulean, SCFP3A, mTurquoise, monomeric Midoriishi- Cyan, TagCFP, mTFP1, EGFP, Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, EYFP, Citrine, Venus, SYFP2, TagYFP, Monomeric Kusabira- Orange, mKOκ, mKO2, mOrange, mOrange2, mRaspberry, mCherry, dsRed, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, mPlum, HcRed-Tandem, mKate2, mNeptune, NirFP, TagRFP657, IFP1.4, iRFP, mKeima Red, LSS-mKate1, LSS- mKate2, PA-GFP, PAmCherryl, PATagRFP, Kaede (green), Kaede (red), KikGR1 (green), KikGR1 (red), PS-CFP2, PS-CFP2, mEos2 (green), mEos2 (red), PSmOrange, or Dronpa.

[0075] In any and all embodiments of the methods disclosed herein, the recombination system comprises a 5’-3’ exonuclease, a DNA polymerase, and a DNA ligase. In any of the preceding embodiments of the methods disclosed herein, the donor heterologous nucleic acid comprises an open reading frame that encodes a bioluminescent protein, a fluorescent protein, a chemiluminescent protein, a heterologous bactericidal gene, a phage protein that modifies host range, or any combination thereof. Additionally or alternatively, in some embodiments, the phage protein that modifies host range is a tail spike protein (e.g., gp11, gp12, and gp17) or a structural phage virion protein that is involved with bacterial cell attachment or degradation of bacterial cell wall components.

[0076] In some embodiments, the encoded gene product(s) produces a detectable signal upon exposure to the appropriate stimuli, and the resulting signal permits detection of bacterial host cells infected by the recombinant phage. In certain embodiments, the open reading frame encodes a protein that serves as a marker that can be identified by screening bacterial host cells infected by a recombinant phage comprising a heterologous nucleic acid sequence comprising the open reading frame. Examples of such markers include by way of example and without limitation: a fluorescent label, a luminescent label, a chemiluminescence label, or an enzymatic label. In some embodiments, the heterologous nucleic acid sequence further comprises sequences naturally found in the bacteriophage, but placed at a non-normally occurring location in the genome.

[0077] In certain embodiments, the open reading frame of the donor heterologous nucleic acid is operably linked to an expression control sequence that is capable of directing expression of the bioluminescent protein, the fluorescent protein, the chemiluminescent protein, the heterologous bactericidal gene, a phage protein that modifies host range, or any combination thereof. For example, the open reading frame may be inserted into the phage genome downstream of or in the 24 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 place of an endogenous phage open reading frame sequence. In some embodiments, the expression control sequence is an inducible promoter or a constitutive promoter (e.g., sarA promoter or lpp promoter). See e.g., Djordjevic & Klaenhammer, Methods in Cell Science 20(1):119-126 (1998). The inducible promoter or constitutive promoter may be an endogenous phage promoter sequence, a non-endogenous phage promoter sequence, or a bacterial host promoter sequence. Additionally or alternatively, in some embodiments, the inducible promoter is a pH-sensitive promoter, or a temperature sensitive promoter.

[0078] In some embodiments, the heterologous nucleic acid sequence is inserted into the phage genome with no loss of endogenous phage genomic sequence. In some embodiments, the heterologous nucleic acid sequence replaces an endogenous phage genomic sequence. In some embodiments, the heterologous nucleic acid sequence includes an endogenous phage genomic sequence that was previously excised from the phage genome.

[0079] In certain embodiments, the heterologous nucleic acid sequence replaces an endogenous phage genomic sequence that is less than the length of the heterologous nucleic acid sequence. Accordingly, in some embodiments, the length of the recombinant phage genome is longer than the length of the wild-type phage genome. In some embodiments, the heterologous nucleic acid sequence replaces an endogenous phage genomic sequence that is greater than the length of the heterologous nucleic acid sequence. Thus, in some embodiments, the length of the recombinant phage genome is shorter than the length of the wild-type phage genome. In certain embodiments, the heterologous nucleic acid sequence replaces an endogenous phage genomic sequence that is equal to the length of the heterologous nucleic acid sequence.

[0080] In some embodiments of the methods disclosed herein, the length of the heterologous nucleic acid sequence is at least 100 bases, at least 200 bases, at least 300 bases, at least 400 bases, at least 500 bases, at least 600 bases, at least 700 bases, at least 800 bases, at least 900 bases, at least 1 kilobase (kb), at least 1.1 kb, at least 1.2 kb, at least 1.3 kb, at least 1.4 kb, at least 1.5 kb, at least 1.6 kb, at least 1.7 kb, at least 1.8 kb, at least 1.9 kb, at least 2.0 kb, at least 2.1 kb, at least 2.2 kb, at least 2.3 kb, at least 2.4 kb, at least 2.5 kb, at least 2.6 kb, at least 2.7 kb, at least 2.8 kb, at least 2.9 kb, at least 3.0 kb, at least 3.1 kb, at least 3.2 kb, at least 3.3 kb, at least 3.4 kb, at least 3.5 kb, at least 3.6 kb, at least 3.7 kb, at least 3.8 kb, at least 3.9 kb, at least 4.0 kb, at least 4.5 kb, at least 5.0 kb, at least 5.5 kb, at least 6.0 kb, at least 6.5 kb, at least 7.0 kb, at least 7.5 kb, at least 8.0 kb, at least 8.5 kb, at least 9.0 kb, at least 9.5 kb, at least 10 kb, or more. In certain embodiments, the heterologous nucleic acid sequence comprises a length that is less than or equal to a length selected from the group consisting of 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, and 10 kb. In some embodiments, the heterologous nucleic acid sequence 25 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 comprises a length that is less than or equal to the maximum length of heterologous nucleic acid sequence that can be packaged into a phage particle comprising the phage genome.

[0081] In some embodiments, the length of the heterologous nucleic acid sequence is from 100 to 500 bases, from 200 to 1,000 bases, from 500 to 1,000 bases, from 500 to 1,500 bases, from 1 kb to 2 kb, from 1.5 kb to 2.5 kb, from 2.0 kb to 3.0 kb, from 2.5 kb to 3.5 kb, from 3.0 kb to 4.0 kb, from 3.5 kb to 4.5 kb, from 4.0 kb to 5.0 kb, from 4.5 kb to 5.5 kb, from 5.0 kb to 6.0 kb, from 5.5 kb to 6.5 kb, from 6.0 kb to 7.0 kb, from 6.5 kb to 7.5 kb, from 7.0 kb to 8.0 kb, from 7.5 kb to 8.5 kb, from 8.0 kb to 9.0 kb, from 8.5 kb to 9.5 kb, or from 9.0 kb to 10.0 kb. Kits

[0082] The present technology provides kits for preparing donor bacterial host cell lysates for cell-free bacteriophage synthesis (CFBS). In one aspect, the present disclosure provides a kit comprising a modified bacterial cell lysate composition comprising a mixture of an E. coli lysate and at least one additional bacterial cell lysate obtained from a bacterial host species that is not E. coli. The at least one additional bacterial cell lysate may be obtained from a bacterial host species selected from among Bacillus species, Klebsiella species, Pseudomonas species, Acinobacter species, Staphylococcus species, Shigella species, Xanthomonas species, Serratia species, Erwinia species, Ralstonia species, Candidatus Liberibacter species, Mycobacterium species, Escherichia species, Enterococcus species, Enterobacter species, Streptococcus species, Flavobacterium species, Borrelia species, Clostridioides species, Helicobacter species, Salmonella species, Clavibacter species, Campylobacter species, Agrobacterium species, Pectobacterium species, Burkholderia species, Xylella species, Yersinia species, Neisseria species, and Listertia species. In some embodiments, the mixture comprises additional bacterial cell lysates obtained from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 distinct bacterial host species that are not E. coli.

[0083] In another aspect, the present disclosure provides a kit comprising a modified bacterial cell lysate composition comprising a mixture of an E. coli lysate and genomic DNA derived from a bacterial host species that is not E. coli. The genomic DNA may be obtained from a bacterial host species may be selected from among Bacillus species, Klebsiella species, Pseudomonas species, Acinobacter species, Staphylococcus species, Shigella species, Xanthomonas species, Serratia species, Erwinia species, Ralstonia species, Candidatus Liberibacter species, Mycobacterium species, Escherichia species, Enterococcus species, Enterobacter species, Streptococcus species, Flavobacterium species, Borrelia species, Clostridioides species, Helicobacter species, Salmonella species, Clavibacter species, 26 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 Campylobacter species, Agrobacterium species, Pectobacterium species, Burkholderia species, Xylella species, Yersinia species, Neisseria species, and Listertia species.

[0084] In any and all embodiments of the kits disclosed herein, the kits further comprise one or more of polyethylene glycol (PEG) polymers (e.g., PEG-8000, PEG-6000), deoxynucleotide triphosphates (dNTPs), stabilizers, and an energy buffer comprising canonical amino acids, phosphoenol pyruvate (PEP), nucleoside triphosphates (NTPs), cofactors, and coenzymes.

[0085] Additionally or alternatively, in some embodiments, the kits further comprise a vector including a nucleic acid sequence encoding a reporter gene, wherein the nucleic acid sequence encoding the reporter gene is operably linked to a promoter that is responsive to a phage RNA polymerase (RNAP), such as SP6 RNA Polymerase, T3 RNA Polymerase and T7 RNA polymerase. In some embodiments, the reporter gene encodes a bioluminescent protein, a fluorescent protein, or a chemiluminescent protein. Examples of bioluminescent proteins include, but are not limited to, Aequorin, firefly luciferase, Renilla luciferase, red luciferase, luxAB, or nanoluciferase. Examples of chemiluminescent proteins include, but are not limited to, β-galactosidase, horseradish peroxidase (HRP), or alkaline phosphatase. Examples of fluorescent proteins include, but are not limited to, sfGFP, TagBFP, Azurite, EBFP2, mKalamal, Sirius, Sapphire, T-Sapphire, ECFP, Cerulean, SCFP3A, mTurquoise, monomeric Midoriishi- Cyan, TagCFP, mTFP1, EGFP, Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, EYFP, Citrine, Venus, SYFP2, TagYFP, Monomeric Kusabira- Orange, mKOκ, mKO2, mOrange, mOrange2, mRaspberry, mCherry, dsRed, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, mPlum, HcRed-Tandem, mKate2, mNeptune, NirFP, TagRFP657, IFP1.4, iRFP, mKeima Red, LSS-mKate1, LSS- mKate2, PA-GFP, PAmCherryl, PATagRFP, Kaede (green), Kaede (red), KikGR1 (green), KikGR1 (red), PS-CFP2, PS-CFP2, mEos2 (green), mEos2 (red), PSmOrange, or Dronpa.

[0086] In any of the preceding embodiments, the kits of the present technology comprise one or more coded / labeled vials that contain a plurality of bacteriophage genomes. The plurality of bacteriophage genomes may be obtained from naturally occurring bacteriophage or genetically engineered bacteriophage. In some embodiments, each coded / labeled vial containing a plurality of bacteriophage genomes corresponds to a different bacteriophage type. In other embodiments, each coded / labeled vial containing a plurality of bacteriophage genomes corresponds to the same bacteriophage type. In some embodiments, each phage vial is assigned a unique code that identifies the bacteriophage in the phage vial, or the types of bacteria that the bacteriophage strain infects. The unique code can be encoded by a machine discernible pattern, such as a bar code, a QR code, an alphanumeric string, or any other pattern that can be discerned by a reader. 27 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 Each unique code may be shown as, for example, a bar code sticker on a vial or container storing a corresponding phage sample. In some embodiments, the kit is stored under conditions that permit the preservation of the bacteriophage genomes for extended periods, such as under bacteriophage-specific, controlled temperature, moisture, and pH conditions.

[0087] The kit may further comprise one or more of: wash buffers and / or reagents, hybridization buffers and / or reagents, labeling buffers and / or reagents, and detection means. The buffers and / or reagents are usually optimized for the particular detection technique for which the kit is intended. Protocols for using these buffers and reagents for performing different steps of the procedure may also be included in the kit. Further optional components of the kits may include expression media for reporter gene products disclosed herein, such as a medium containing nutrients and cofactors for bioluminescence, devices such as a lamp configured to illuminate at specific wavelengths of light to detect biofluorescence, and devices for measuring the extent of expression of the reporter genes, such as a photometer or photodetector.

[0088] In some embodiments, the kits further comprise positive control heterologous nucleic acid sequences to correct for any variability in the CFBS systems between experimental runs. The kits may also comprise instructions for use, software for automated analysis, containers, packages such as packaging intended for commercial sale and the like. EXAMPLES Example 1: Non-Model Bacteria as TXTL Donors

[0089] E. coli has historically been the TXTL donor of choice not because it inherently a more productive background for protein expression, but because it has benefiting from 60+ years of sustained research and development of ever improving molecular biology and analytical tools that simplify genetic and metabolic manipulation of this thoroughly investigated model bacterium. The last ~20 years have shown there are advantages to establishing novel CFES from diverse bacterial backgrounds to benefit from unique metabolisms and physicochemical environments. FIGs.6-7 summarize all non-model (re: non-E. coli) coupled transcription / translation CFES reported in peer-reviewed publications for which reporter protein (usually green fluorescent protein (GFP) a derivative) yields are given in readily comparable units (molarity or mass concentration).

[0090] Advantage 1: TXTL background favors synthesis of soluble (re: functional) endogenous proteins. This was phenomenon was most clearly illustrated in expression of high GC% genes in Streptomyces TXTL cell-free systems. Streptomyces protein TbrP expressed by Streptomyces TXTL achieved a comparable yield to E. coli TXTL expression (17 vs 23.9 28 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 µg / mL) but the soluble TbrP fractions were 70.4% and 9.6% respectively1. The differences in solubility relate to proper protein folding likely influenced by the presence and relative concentrations of protein-folding chaperones present in Streptomyces lysates, but not E. coli.

[0091] Advantage 2: Non-model CFES can be used to screen for and optimize gene expression using endogenous transcription regulatory elements. The physicochemical environment (i.e. optimal cations concentrations, molecular crowding, incubation temperatures) in CFES influences the folding of proteins as well as DNA and RNA2. The secondary structure of DNA plays a role in the promoter and terminator strength; likewise, secondary structure of mRNAs can influence translation rates and RNA stability (re: longevity)3. CFES mimicking the physicochemical environment of a non-model bacterial cytoplasm reflect the transcriptional behavior of genetic parts in vivo4. In addition to Advantage 1, diverse bacterial lysates enable prototyping of protein expression systems and metabolic pathways for chemical synthesis in high-throughput as opposed to lower-throughput traditional cloning techniques4-6.

[0092] Advantage 3: Cell-free systems enable the study pathogenic or fastidious organism transcription / translation in a Biosafety Level 1 (BSL1) environment. Recent work involving potential pathogens Yersinia pestis, Klebsiella pneumoniae, and Cutibacterium acnes established the feasibility of protein synthesis lysates derived from each bacteria7-9. Beyond protein synthesis, CFES using Streptococcus and Staphylococcus TXTL machinery have been established to interrogate intracellular mechanisms of antibiotic-resistance against transcription / translation inhibitors10, 11. Additionally, cell-free protein synthesis under aerobic conditions has been demonstrated for microaerophile C. acnes and obligate anaerobe Clostridioides autoethanogenum, offering CFES as a simplified approach for genetic part, protein, and pathway engineering for fastidious organisms under aerobic conditions6, 8.

[0093] Advantage 4: Bacterial diversity suggests potential for high-yield protein expression. Rapidly-growing Vibrio natriegens has recently been proposed as an alternative TXTL donor to E. coli for high-yield CFPS12-14. While V. natriegens CFES have yet to match E. coli batch-mode CFPS yields, V. natriegens systems have improved from 0.26 mg / mL in 2018 to >1.8 mg / mL in 2023 through standard reaction condition and process development workflows12, 15. Note: In 2017, the max batch-mode E. coli CFPS yield was 2.3 mg / mL and has increased to ~4 mg / mL (2021)16, 17. We anticipate that further process improvements and strain engineering will enable non-model bacteria to match or overtake E. coli CFPS.

[0094] Advantage 5: An additional advantage of using non-model TXTL is their expression of endogenous gene clusters for chemical synthesis without the need for extensive genetic 29 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 engineering. Multi-enzyme biosynthesis pathways impose a severe metabolic burden on E. coli when multiple genes must be co-expressed concurrently. A workaround is to express individual pathway genes in different E. coli cell lines then to pool the lysates for cell-free synthesis of high-value chemicals from a reconstituted pathway18. Expression systems using TXTL already carrying desired biosynthesis pathways can avoid relatively complicated strategies for chemical synthesis.

[0095] As seen from the rapid improvement in V. natriegens CFES protein synthesis yields (above), standard optimization workflows for improving cell-free expression systems are a viable approach to improve yields perhaps to parity with those in E. coli-based systems. These workflows include optimization of lysate preparation (e.g. culture medium, growth temperature, harvest OD, lysis method), post-lysis processing (runoff reactions or dialysis), reaction condition optimization (e.g. temperature, [Mg2+] and [K+], template concentration, lysate percentage (v / v) in reactions, supplementation with protease or RNase inhibitors, endogenous or exogenous transcription, etc.), and strain engineering9, 14, 19-21.

[0096] Some considerations worth noting are the availability of chassis strains for certain genera or species with suitable metabolism, the identification of positive and negative effector genes for strain engineering, and the overall knowledge base supporting a potential non-model TXTL donor. For example establishing an Acinetobacter baumannii CFES would be challenging because Acinetobacter species often lack several enzymes to complete canonical glycolysis pathway (glucose ^ ^pyruvate), which provide sustained ATP in modern CFES22, 23. The gaps in glycolysis could be filled by plasmid-based expression of the missing enzymes in Acintobacter prior to lysis or exogenously adding purified enzymes to CFES reactions. Alternatively, a chassis strain may be engineered for chromosomal expression of missing enzymes. For example, A. baumannii relative A. baylyi ADP1 only lacks one glycolytic enzyme, pyruvate kinase (pykF), and has been engineered for glucose utilization24. Beyond metabolism, there may be strain characteristics that make them challenging to physically process to create lysates. Yang et al. (2022) demonstrated that deleting genes associated with exopolysaccharide capsule expression in Klebsiella pneumoniae facilitated centrifugation of live cells by eliminating the native mucoid Klebsiella phenotype, which ultimately allowed creation of a productive Klebsiella CFES9. Similar metabolic or phenotypic changes are surely feasible for other non-model organisms for which genetic engineering tools have been developed.

[0097] Non-model CFES have expanded in diversity and productivity, especially in the last 10 years. In most cases, standard CFES optimization workflows have resulted in sustained improvements in CFES yields and variety of protein or chemical products. Some intractable 30 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 organisms may be engineered for ease of use or to establish minimum metabolic function consistent with standard CFES processes and reagents. Multi-omic Interrogation of especially low-yield CFES may reveal bottlenecks and indicate appropriate direction of optimization efforts. TXTL chassis strain engineering should be preceded by screening of potential positive and negative effectors to avoid unfocused effort. Rather than by strain engineering, effector screening can be accelerated by using a single lysate configuration supplemented with potential effector proteins generated by in parallel CFPS from plasmid templates, another example of the positive feedback of improved CFES25. Advancement of diverse CFES will continue to enrich the understanding of intracellular processes, genetic tools, and complex biochemistries.

[0098] CFBS has great potential as a tool to study phage-bacteria interactions, therapeutic phage manufacturing, and phage engineering. Historically, investigation of interactions between phage and bacteria stopped at the cell membrane unless the bacteria was a permissive host. Here, “permissive” indicates that the phage is able to deliver its genome into the bacteria and the bacterial TXTL machinery is able to replicate new functional phage particles in contrast to an unproductive infection in which phage genomes enter a cell, but new viable phage are not produced. Phage host-specificity implies that most phage receptor binding proteins do not interact with most bacterial surface receptors. In other words, it is difficult to differentiate between phage-bacteria interactions in which infection fails (phage genomes never enter the cell) or if infections are unproductive (phage-mediated transcription, translation, genome replication, or genome packaging and capsid assembly fail). The elimination of the barrier of cell-walls and membranes in CFBS allow for the investigation of the fate of phage genomes in potentially non- permissive or low efficiency hosts and to diagnose at which step in the phage replication process fails. This knowledge could be useful in understanding bacterial population dynamics in nature and in microbiomes. In such scenarios, cell-free systems are ideal, especially if bacterial populations in question uncultured (e.g. in metagenomes) or are difficult to transfect with phage DNA.

[0099] CFBS is useful because its components are relatively stable and can be stored long- term (at least 1y) without loss of activity and phage can be produced at therapeutic titers within 8h. Endotoxin-free cell-lysates can be prepared in advance, frozen, and thawed when needed, along with other reaction reagents. “Just-add-water” lyophilized CFES, include for point-of-care therapeutics has also been demonstrated feasible. Notably, DNA is highly stable when lyophilized with a projected half-life of ~100y stored in darkness at room temperature in a controlled humidity environment32. While, to our knowledge, lyophilized CFBS components 31 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 have not been reconstituted from lyophilized stocks, prior work suggests it is feasible and would enable for point-of-care phage production without expensive cold-chain requirements33.

[0100] Most phage engineering methods require well-characterized and highly-competent hosts. Common phage engineering relies on homologous recombination of phage DNA while within a living bacterium isolating engineered phage from a mixed population of wild-type and modified phage by plaque assay34. The primary limitation of these methods is that the recombination host is either permissive to phage infection (i.e. DNA entry through infection) or competent for transformation (amenable to naked DNA entry through heat-shock or electroporation). Competence is required for infection-based methods as well to provide a recombination template for the phage DNA34. Furthermore, recombination methods have <100% efficiency, meaning that isolating phage mutants from resulting progeny may require screening of 100s of plaques or repeated enrichment steps. More recent phage engineering methods utilize assembly of synthetic DNA fragments with modified sequences generated by DNA synthesis or high-fidelity PCR followed by transformation into a competent host35-37. The advantage of these methods is that theoretically 100% of progeny phage will be the desired mutant since no wild- type DNA is included in the transformation step. The requirement for competent hosts to recombine or reboot phage generally limits phage engineering candidates to those with well- characterized hosts with high-efficiency transformation protocols. CFBS for phage engineering could enable rapid all-in vitro workflows that could be generalized to diverse phage. FIG.2 illustrates verified and speculative approaches to CFBS implementation beyond well established model phage and bacteria. Example 2: CFBS Methods of the Present Technology

[0101] We aim to propagate the phage in the table below using non-standard cell-free bacteriophage synthesis (CFBS) reactions. The general method for CFBS is described below. Preparation methods for non-standard CFBS compositions to produce diverse phage are also described below. We anticipate that at least one of the non-standard approaches will be required for each phage / host-pair. Success of CFBS reactions will be determined by plaque assays of CFBS reaction products infecting appropriate permissive hosts.32 4859-9106-8900.1Atty. Dkt. No.: 136669-0119

[0102] It is also anticipated that the modified bacterial cell lysate compositions of the present technology will promote high-yield protein expression and high titers (e.g., 107PFU / ml) of at least one of the above fastidious or pathogenic test phages (e.g., gh-1, vB_KpnP-Michel, SPP1).

[0103] Reagents: Cell-free expression system / cell-free bacteriophage synthesis (CFES / CFBS) components:, NTPs (ATP, CTP, GTP, UTP), dNTPs (dATP, dGTP, dCTP, dTTP), E. coli total tRNA mixture (from strain MRE600), 5-Formyl-5,6,7,8-tetrahydrofolic acid (folinic acid), 4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), nicotinamide adenine dinucleotide (NAD), cyclic adenosine monophosphate (cAMP), coenzyme A (CoA), spermidine, 18 canonical amino acids excluding glutamate and leucine, polyethylene glycol 8000 (PEG-8000), maltodextrin, sodium oxalate, 1,4-dithiothreitol (DTT), potassium glutamate (K(glu)), magnesium glutamate (Mg(glu)2), magnesium sulfate, calcium chloride, DNase I, lysozyme, and proteinase K. Leucine, phosphoenolpyruvate (PEP), 3-phosphoglycerate (3-PGA)UTP from Alfa Aesar. RecBCD nuclease inhibitor GamS. LB medium (tryptone, yeast extract, sodium chloride. Phosphate buffer (potassium phosphate monobasic, potassium phosphate dibasic).

[0104] Cell-lysate preparation: Cell-extracts were prepared based on Kwon et al. (2019) with modification. E. coli BL21 was grown overnight in 1 mL LB, supplemented with appropriate antibiotics, inoculated by a single isolated colony. Overnight cultures were diluted 1:10 in prewarmed selective LB and incubated for 1h at 37˚C to bring the cultures to log-phase. Next, cultures were diluted 1:100 in pre- warmed 2×YTP media (16 g / L of tryptone, 10 g / L of yeast extract, 5 g / L of sodium chloride, 7 g / L of potassium phosphate dibasic, 3 g / L of potassium phosphate monobasic, pH 7.2) plus appropriate antibiotics in 500 mL baffled-flasks and incubated at 37˚C at 250 rpm and growth monitored by OD600. Nutrient Broth (NB), brain-heart infusion (BHI), Terrific Broth (TB), or other rich medium may be used as alternatives to 2xYTP. When OD600 reached 2.0-3.0, flasks were immediately placed on ice and gently mixed to quickly bring down temperature. Samples were kept cold from this step onward. After 15 min on ice, each culture was centrifuge at 10,000 x g for 5 min at 4˚C. The supernatants were removed, and pellets washed three times with ice-cold S30B buffer (10 mM Tris-Cl, pH 8.2, 14 mM magnesium glutamate, 60 mM potassium glutamate, 2 mM DTT). Pellet wet-mass (g) was recorded followed by flash-freezing in liquid nitrogen and storage at -80˚C overnight. Frozen pellets were thawed slowly in ice- water then resuspended in S30B (0.8 mL per g wet-mass) and transferred to 1.5 mL microcentrifuge tubes with final volumes 500-1000 μL. Cell slurries were lysed in an ice-water bath using the Qsonica Q125 Sonicator with 1 / 8” probe set to 50% amplitude with 10s on / off pulses to avoid overheating samples. Target total energy input was calculated using the following formula33:(3)^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^(^^^^)=^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ^^^^ ( ^^^^ ^^^^)−33.61.833 4859-9106-8900.1Atty. Dkt. No.: 136669-0119

[0105] DTT was added to each lysate for a final 3 mM concentration. Cell-lysates were centrifuged at 12,000 x g for 10 min at 4˚C. Supernatants from each strain were consolidated, then incubated at 37˚C shaking for 80 min for a runoff reaction followed by another centrifugation at 10,000 x g for 10 min at 4˚C. Finally, these clarified supernatants were aliquoted and flash-frozen with liquid nitrogen then stored at -80˚C. Presence of residual bacteria was checked by spotting clarified lysates on LB agar and incubating overnight at 37˚C.

[0106] Standard Cell-free bacteriophage synthesis reaction setup: Bacteriophage synthesis was performed in 15 μL reactions incubated at 30˚C in 1.5 mL tubes with cell extracts occupying 33% volume and reaction buffer and phage genomic DNA (gDNA) templates the remaining 67%. Reactions contained 57 mM HEPES, pH 8, 0-300 mM K(glu), 0-30 mM Mg(glu)2, 0.4 mM NAD, 0.27 mM CoA, 0.75 mM cAMP, 2 mM spermidine, 0-4 mM DTT, 1.5 mM ATP and GTP, 0.9 mM CTP and UTP, 0.2 mg / mL E. coli tRNA, 0.068 mM folinic acid, 1-2 mM of each canonical amino acid except glutamate, 33 mM PEP, 12.66 mg / mL maltodextrin, 0.1-10 nM phage gDNA.30 mM 3-PGA may be used in place of PEP. Reactions are supplemented with 0-2 mM dNTPs and 0-4% PEG-8000. Reaction components were mixed with DNA added last then kept on ice for 5 min prior to incubation at 30˚C for 4-20h.

[0107] Hybrid lysate configurations: FIG.2A: Rebooting phage with standardized E. coli- TXTL from phage genomes (gDNA); FIG.2B: Establish a standard Gram (-) TXTL (e.g. E. coli BL21) and equivalent high-productivity Gram (+) TXTL (e.g. B. megaterium) for rebooting corresponding Gram (-) and (+) phage respectively; FIG.2C: Standardized E. coli-TXTL supplemented with pre-identified essential host protein (co-expressed or exogenous); FIG.2D: Standardized E. coli-TXTL supplemented with non-E. coli cell-lysate to provide non- preidentified host factors to reboot non-coliphage; FIG.2E: representation of diverse bacteria specifically targeted by diverse phage; FIG.2F: Establish a Genus-level CFBS ; FIG.2G: “Universal” CFBS comprised of a mixture of diverse cell-lysates; FIG.2H: in vitro phage self- assembly by mixing phage gDNA with pre-expressed capsid protein in buffer + ATP; FIG.2I: CFBS by decoupled transcription (TX) and translation (TL) by generating phage mRNAs through in vitro transcription to then be added to standardized cell-extracts for translation and phage assembly; FIG.2J: Establish a cell-free mimic proteome to phage host to provide essential host factors by (1) cell-free expression of the host proteome directed by its whole genome then (2) rebooting phage from its gDNA.

[0108] Optimization of CFBS Reactions: CFBS reaction conditions may be optimized for phage yield with respect to the following factors: bacterial growth conditions (media and growth temperature), sonication lysis energy input, runoff reactions, reaction composition (buffer pH 34 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 and concentrations of K(glu), Mg(glu)2,DTT, PEG-8000, phage DNA, cell lysate, nuclease- inhibitors, protease-inhibitors, RNase-inhibitors, or other reaction enhancers or supplements), and reaction conditions (e.g. reaction volume, aerobic / anaerobic condition, incubation temperature, and incubation time).

[0109] CFBS Approaches of the Present Technology:

[0110] Non-E. coli Bacteria list: Shigella, Xanthomonas, Serratia, Erwinia, Ralstonia, Candidatus Liberibacter, Mycobacterium, Escherichia, Staphylococcus, Klebsiella, Acinetobacter, Pseudomonas, Enterococcus, Enterobacter, Streptococcus, Flavobacterium, Borrelia, Clostridioides, Helicobacter, Salmonella, Clavibacter, Campylobacter, Agrobacterium, Pectobacterium, Burkholderia, Xylella, Yersinia, Neisseria, Listertia, Bacillus.

[0111] FIG.2B: Standard Gram (-) TXTL (e.g. E. coli BL21) and equivalent Standard high- productivity Gram (+) TXTL (e.g. Bacillus megaterium or Streptomyces) for rebooting Gram (-) and (+) phage respectively. Synthesize Gram (+) phage using standard TXTL from Gram (+) bacterial lysates. Lysate preparation methods would be like description above. The Gram (+) lysate CFBS reaction conditions may be optimized as described above.

[0112] FIG.2D: Hybrid cell-lysates comprised of standard E. TXTL supplemented withany non-E. coli cell-lysate from bacteria described herein. This provides host factors and physicochemical conditions to simulate host bacteria driven by the productivity established E. coli-TXTL CFBS. Hybrid lysates may be prepared using different proportions of non-E. coli lysate for example 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 10%-99%. Hybrid lysate CFBS reaction conditions may be optimized as described above.

[0113] FIG.2F: Non-model lysate-based TXTL CFBS Reactions. Prepare cell lysates relatives of host bacteria at the Genus level. For example, lysates may be derived from and not limited to the following genera: Shigella, Xanthomonas, Serratia, Erwinia, Ralstonia, Candidatus Liberibacter, Mycobacterium, Escherichia, Staphylococcus, Klebsiella, Acinetobacter, Pseudomonas, Enterococcus, Enterobacter, Streptococcus, Flavobacterium, Borrelia, Clostridioides, Helicobacter, Salmonella, Clavibacter, Campylobacter, Agrobacterium, Pectobacterium, Burkholderia, Xylella, Yersinia, Neisseria, Listeria, Bacillus, Nocardia, Chlamydia. CFBS reaction conditions may be optimized as described above.

[0114] FIG.2G: “Universal” CFBS comprised of a mixture of cell lysates including 2-10 cell- lysates derived from different non-E. coli strains of bacteria. Diverse lysates contribute host factors to enable synthesis of diverse phage by CFBS without elucidating “essential” host factors. Universal CFBS reactions may or may not include an E. coli lysate component. 35 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 Universal CFBS lysates may be prepared using different proportions of non-E. coli lysate for example 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 10%-99%. Universal CFBS reaction conditions may be optimized as described above.

[0115] FIG.2J: CFBS supplemented with synthetic host proteome. Establish a cell-free mimic proteome to phage host to provide essential host factors by (1) cell-free expression of the host proteome directed by its whole genome then (2) rebooting phage from its gDNA. First, a cell- free reaction is prepared for CFBS omitting phage DNA. Instead, the reaction is initiated by adding purified host bacteria gDNA to recapitulate the host proteome in situ (See IGeTT method). This iGeTT reaction will be carried out for 0-8h prior to addition of phage DNA to transition to phage synthesis. Alternatively, a CFBS reaction can be prepared as normal but supplemented with a portion of a separate iGeTT reaction directed host DNA. CFBS supplemented with synthetic host proteome maybe supplemented using different proportions of iGeTT reactions containing host gDNA: 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 10%-99%. EQUIVALENTS

[0116] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the present technology. It is to be understood that this present technology is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0117] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0118] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range 36 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

[0119] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification. REFERENCES 1. Li J, Wang H, Kwon YC, Jewett MC. Establishing a high yielding streptomyces-based cell-free protein synthesis system. Biotechnol Bioeng.2017;114(6):1343-1353. Epub 2017 / 01 / 24. doi: 10.1002 / bit.26253. Available from:2. Ge X, Luo D, Xu J. Cell-free protein expression under macromolecular crowding conditions. PLoS One.2011;6(12):e28707. Epub 2011 / 12 / 17. doi: 10.1371 / journal.pone.0028707. Available from:3. van Kooten M, Scheidegger CA, Christen M, Christen B. The transcriptional landscape of a rewritten bacterial genome reveals control elements and genome design principles. Nat Commun.2021;12(1):3053. Epub 2021 / 05 / 26. doi: 10.1038 / s41467-021-23362-y. Available from: https: / / www.ncbi.nlm.nih.gov / pubmed / 34031412. 4. Yim SS, Johns NI, Park J, Gomes AL, McBee RM, Richardson M, Ronda C, Chen SP, Garenne D, Noireaux V, Wang HH. Multiplex transcriptional characterizations across diverse bacterial species using cell-free systems. Mol Syst Biol.2019;15(8):e8875. Epub 2019 / 08 / 30. doi: 10.15252 / msb.20198875. Available from: https: / / www.ncbi.nlm.nih.gov / pubmed / 31464371. 5. Moore SJ, Lai HE, Chee SM, Toh M, Coode S, Chengan K, Capel P, Corre C, de Los Santos EL, Freemont PS. A Streptomyces venezuelae Cell-Free Toolkit for Synthetic Biology. ACS Synth Biol.2021;10(2):402-411. Epub 2021 / 01 / 27. doi: 10.1021 / acssynbio.0c00581. Available from:37 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 6. Kruger A, Mueller AP, Rybnicky GA, Engle NL, Yang ZK, Tschaplinski TJ, Simpson SD, Kopke M, Jewett MC. Development of a clostridia-based cell-free system for prototyping genetic parts and metabolic pathways. Metab Eng.2020;62:95-105. Epub 2020 / 06 / 17. doi: 10.1016 / j.ymben.2020.06.004. Available from:7. McDonald ND, Rhea KA, Berk KL, Zacharko JL, Miklos AE. Cell-Free Protein Systems from Yersinia pestis are Functional and Growth-Temperature Dependent. ACS Synth Biol. 2021;10(12):3604-3607. Epub 2021 / 12 / 03. doi: 10.1021 / acssynbio.1c00505. Available from:8. Fabrega MJ, Knodlseder N, Nevot G, Sanvicente M, Toloza L, Santos-Moreno J, Guell M. Establishing a Cell-Free Transcription-Translation Platform for Cutibacterium acnes to Prototype Engineered Metabolic and Synthetic Biology. ACS Biomater Sci Eng.2021. Epub 2022 / 01 / 01. doi: 10.1021 / acsbiomaterials.1c00894. Available from:9. Yang C, Yang M, Zhao W, Ding Y, Wang Y, Li J. Establishing a Klebsiella pneumoniae- Based Cell-Free Protein Synthesis System. Molecules.2022;27(15). Epub 2022 / 07 / 29. doi: 10.3390 / molecules27154684. Available from: https: / / www.ncbi.nlm.nih.gov / pubmed / 35897861. 10. Dandliker PJ, Pratt SD, Nilius AM, Black-Schaefer C, Ruan X, Towne DL, Clark RF, Englund EE, Wagner R, Weitzberg M, Chovan LE, Hickman RK, Daly MM, Kakavas S, Zhong P, Cao Z, David CA, Xuei X, Lerner CG, Soni NB, Bui M, Shen LL, Cai Y, Merta PJ, Saiki AY, Beutel BA. Novel antibacterial class. Antimicrob Agents Chemother.2003;47(12):3831-3839. Epub 2003 / 11 / 26. doi: 10.1128 / AAC.47.12.3831-3839.2003. Available from:11. Murray RW, Melchior EP, Hagadorn JC, Marotti KR. Staphylococcus aureus cell extract transcription-translation assay: firefly luciferase reporter system for evaluating protein translation inhibitors. Antimicrob Agents Chemother.2001;45(6):1900-1904. Epub 2001 / 05 / 17. doi: 10.1128 / AAC.45.6.1900-1904.2001. Available from:12. Wiegand DJ, Lee HH, Ostrov N, Church GM. Establishing a Cell-Free Vibrio natriegens Expression System. ACS Synth Biol.2018;7(10):2475-2479. Epub 2018 / 08 / 31. doi: 10.1021 / acssynbio.8b00222. Available from: https: / / www.ncbi.nlm.nih.gov / pubmed / 30160938. 38 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 13. Failmezger J, Scholz S, Blombach B, Siemann-Herzberg M. Cell-Free Protein Synthesis From Fast-Growing Vibrio natriegens. Front Microbiol.2018;9:1146. Epub 2018 / 06 / 19. doi: 10.3389 / fmicb.2018.01146. Available from: https: / / www.ncbi.nlm.nih.gov / pubmed / 29910785. 14. Des Soye BJ, Davidson SR, Weinstock MT, Gibson DG, Jewett MC. Establishing a High-Yielding Cell-Free Protein Synthesis Platform Derived from Vibrio natriegens. ACS Synth Biol.2018;7(9):2245-2255. Epub 2018 / 08 / 15. doi: 10.1021 / acssynbio.8b00252. Available from:15. Zhu B, Gan R, Cabezas MD, Kojima T, Nicol R, Jewett MC, Nakano H. Increasing cell- free gene expression yields from linear templates in Escherichia coli and Vibrio natriegens extracts by using DNA-binding proteins. Biotechnol Bioeng.2020;117(12):3849-3857. Epub 2020 / 08 / 21. doi: 10.1002 / bit.27538. Available from:16. Caschera F, Noireaux V. Synthesis of 2.3 mg / ml of protein with an all Escherichia coli cell-free transcription-translation system. Biochimie.2014;99:162-168. Epub 2013 / 12 / 12. doi: 10.1016 / j.biochi.2013.11.025. Available from:17. Garenne D, Thompson S, Brisson A, Khakimzhan A, Noireaux V. The all-E. coliTXTL toolbox 3.0: new capabilities of a cell-free synthetic biology platform. Synth Biol (Oxf). 2021;6(1):ysab017. Epub 2021 / 10 / 30. doi: 10.1093 / synbio / ysab017. Available from:18. Dudley QM, Nash CJ, Jewett MC. Cell-free biosynthesis of limonene using enzyme- enriched Escherichia coli lysates. Synth Biol (Oxf).2019;4(1):ysz003. Epub 2019 / 03 / 16. doi: 10.1093 / synbio / ysz003. Available from:19. Falgenhauer E, von Schonberg S, Meng C, Muckl A, Vogele K, Emslander Q, Ludwig C, Simmel FC. Evaluation of an E. coli Cell Extract Prepared by Lysozyme-Assisted Sonication via Gene Expression, Phage Assembly and Proteomics. Chembiochem.2021;22(18):2805-2813. Epub 2021 / 07 / 10. doi: 10.1002 / cbic.202100257. Available from:20. Hong SH, Kwon YC, Martin RW, Des Soye BJ, de Paz AM, Swonger KN, Ntai I, Kelleher NL, Jewett MC. Improving cell-free protein synthesis through genome engineering of Escherichia coli lacking release factor 1. Chembiochem.2015;16(5):844-853. Epub 2015 / 03 / 05. doi: 10.1002 / cbic.201402708. Available from: https: / / www.ncbi.nlm.nih.gov / pubmed / 25737329. 39 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 21. Tian R, Wang M, Shi J, Qin X, Guo H, Jia X, Li J, Liu L, Du G, Chen J, Liu Y. Cell-free synthesis system-assisted pathway bottleneck diagnosis and engineering in Bacillus subtilis. Synth Syst Biotechnol.2020;5(3):131-136. Epub 2020 / 07 / 09. doi: 10.1016 / j.synbio.2020.06.006. Available from: https: / / www.ncbi.nlm.nih.gov / pubmed / 32637666. 22. Vallenet D, Nordmann P, Barbe V, Poirel L, Mangenot S, Bataille E, Dossat C, Gas S, Kreimeyer A, Lenoble P, Oztas S, Poulain J, Segurens B, Robert C, Abergel C, Claverie JM, Raoult D, Medigue C, Weissenbach J, Cruveiller S. Comparative analysis of Acinetobacters: three genomes for three lifestyles. PLoS One.2008;3(3):e1805. Epub 2008 / 03 / 20. doi: 10.1371 / journal.pone.0001805. Available from:23. Jewett MC, Swartz JR. Mimicking the Escherichia coli cytoplasmic environment activates long-lived and efficient cell-free protein synthesis. Biotechnol Bioeng.2004;86(1):19- 26. Epub 2004 / 03 / 10. doi: 10.1002 / bit.20026. Available from:24. Kannisto M, Aho T, Karp M, Santala V. Metabolic engineering of Acinetobacter baylyi ADP1 for improved growth on gluconate and glucose. Appl Environ Microbiol. 2014;80(22):7021-7027. Epub 2014 / 09 / 07. doi: 10.1128 / AEM.01837-14. Available from:25. Airen IO. Genome-wide Functional Genomic Analysis for Physiological Investigation and Improvement of Cell-Free Protein Synthesis: Standford University; 2011. 26. Sakai A, Deich CR, Nelissen FHT, Jonker AJ, Bittencourt DMC, Kempes CP, Wise KS, Heus HA, Huck WTS, Adamala KP, Glass JI. Traditional protocols and optimization methods lead to absent expression in a mycoplasma cell-free gene expression platform. Synth Biol (Oxf). 2022;7(1):ysac008. Epub 2022 / 07 / 02. doi: 10.1093 / synbio / ysac008. Available from:27. Failmezger J, Rauter M, Nitschel R, Kraml M, Siemann-Herzberg M. Cell-free protein synthesis from non-growing, stressed Escherichia coli. Sci Rep.2017;7(1):16524. Epub 2017 / 12 / 01. doi: 10.1038 / s41598-017-16767-7. Available from:28. Garenne D, Bowden S, Noireaux V. Cell-free expression and synthesis of viruses and bacteriophages: applications to medicine and nanotechnology. Current Opinion in Systems Biology.2021;28. doi: ARTN 100373 40 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 10.1016 / j.coisb.2021.100373. Available from: <Go to ISI>: / / WOS:000850439600002. 29. Emslander Q, Vogele K, Braun P, Stender J, Willy C, Joppich M, Hammerl JA, Abele M, Meng C, Pichlmair A, Ludwig C, Bugert JJ, Simmel FC, Westmeyer GG. Cell-free production of personalized therapeutic phages targeting multidrug-resistant bacteria. Cell Chem Biol. 2022;29(9):1434-1445 e1437. Epub 2022 / 07 / 13. doi: 10.1016 / j.chembiol.2022.06.003. Available from: https: / / www.ncbi.nlm.nih.gov / pubmed / 35820417. 30. Wurstle S, Stender J, Hammerl JA, Vogele K, Rothe K, Willy C, Bugert JJ. Practical Assessment of an Interdisciplinary Bacteriophage Delivery Pipeline for Personalized Therapy of Gram-Negative Bacterial Infections. Pharmaceuticals (Basel).2022;15(2). Epub 2022 / 02 / 27. doi: 10.3390 / ph15020186. Available from: https: / / www.ncbi.nlm.nih.gov / pubmed / 35215298. 31. Jonczyk E, Klak M, Miedzybrodzki R, Gorski A. The influence of external factors on bacteriophages--review. Folia Microbiol (Praha).2011;56(3):191-200. Epub 2011 / 06 / 01. doi: 10.1007 / s12223-011-0039-8. Available from: https: / / www.ncbi.nlm.nih.gov / pubmed / 21625877. 32. Ivanova NV, Kuzmina ML. Protocols for dry DNA storage and shipment at room temperature. Mol Ecol Resour.2013;13(5):890-898. Epub 2013 / 06 / 25. doi: 10.1111 / 1755- 0998.12134. Available from:33. Wilding KM, Zhao EL, Earl CC, Bundy BC. Thermostable lyoprotectant-enhanced cell- free protein synthesis for on-demand endotoxin-free therapeutic production. N Biotechnol. 2019;53:73-80. Epub 2019 / 07 / 28. doi: 10.1016 / j.nbt.2019.07.004. Available from:34. Kilcher S, Loessner MJ. Engineering Bacteriophages as Versatile Biologics. Trends Microbiol.2019;27(4):355-367. Epub 2018 / 10 / 17. doi: 10.1016 / j.tim.2018.09.006. Available from: https: / / www.ncbi.nlm.nih.gov / pubmed / 30322741. 35. Ando H, Lemire S, Pires DP, Lu TK. Engineering Modular Viral Scaffolds for Targeted Bacterial Population Editing. Cell Syst.2015;1(3):187-196. Epub 2016 / 03 / 15. doi: 10.1016 / j.cels.2015.08.013. Available from: https: / / www.ncbi.nlm.nih.gov / pubmed / 26973885. 36. Mitsunaka S, Yamazaki K, Pramono AK, Ikeuchi M, Kitao T, Ohara N, Kubori T, Nagai H, Ando H. Synthetic engineering and biological containment of bacteriophages. Proc Natl Acad Sci U S A.2022;119(48):e2206739119. Epub 2022 / 11 / 22. doi: 10.1073 / pnas.2206739119. Available from: https: / / www.ncbi.nlm.nih.gov / pubmed / 36409909. 37. Kilcher S, Studer P, Muessner C, Klumpp J, Loessner MJ. Cross-genus rebooting of custom-made, synthetic bacteriophage genomes in L-form bacteria. Proc Natl Acad Sci U S A. 41 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 2018;115(3):567-572. Epub 2018 / 01 / 05. doi: 10.1073 / pnas.1714658115. Available from:38. Kelwick R, Webb AJ, MacDonald JT, Freemont PS. Development of a Bacillus subtilis cell-free transcription-translation system for prototyping regulatory elements. Metab Eng. 2016;38:370-381. Epub 2016 / 10 / 05. doi: 10.1016 / j.ymben.2016.09.008. Available from:39. Moore SJ, MacDonald JT, Wienecke S, Ishwarbhai A, Tsipa A, Aw R, Kylilis N, Bell DJ, McClymont DW, Jensen K, Polizzi KM, Biedendieck R, Freemont PS. Rapid acquisition and model-based analysis of cell-free transcription-translation reactions from nonmodel bacteria. Proc Natl Acad Sci U S A.2018;115(19):E4340-E4349. Epub 2018 / 04 / 19. doi: 10.1073 / pnas.1715806115. Available from: https: / / www.ncbi.nlm.nih.gov / pubmed / 29666238. 40. Zhang L, Lin X, Wang T, Guo W, Lu Y. Development and comparison of cell-free protein synthesis systems derived from typical bacterial chassis. Bioresour Bioprocess. 2021;8(1):58. Epub 2021 / 07 / 13. doi: 10.1186 / s40643-021-00413-2. Available from:41. Wang H, Li J, Jewett MC. Development of a Pseudomonas putida cell-free protein synthesis platform for rapid screening of gene regulatory elements. Synth Biol (Oxf). 2018;3(1):ysy003. Epub 2018 / 05 / 09. doi: 10.1093 / synbio / ysy003. Available from:42. Nakashima N, Tamura T. Cell-free protein synthesis using cell extract of Pseudomonas fluorescens and CspA promoter. Biochem Biophys Res Commun.2004;319(2):671-676. Epub 2004 / 06 / 05. doi: 10.1016 / j.bbrc.2004.05.034. Available from:43. Moore SJ, Lai HE, Needham H, Polizzi KM, Freemont PS. Streptomyces venezuelae TX- TL - a next generation cell-free synthetic biology tool. Biotechnol J.2017;12(4). Epub 2017 / 02 / 01. doi: 10.1002 / biot.201600678. Available from:44. Li J, Wang H, Jewett MC. Expanding the palette of Streptomyces-based cell-free protein synthesis systems with enhanced yields. Biochemical Engineering Journal.2018;130:29-33. doi: 10.1016 / j.bej.2017.11.013. 45. Xu H, Liu WQ, Li J. Translation Related Factors Improve the Productivity of a Streptomyces-Based Cell-Free Protein Synthesis System. ACS Synth Biol.2020;9(5):1221-1224. 42 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 Epub 2020 / 04 / 25. doi: 10.1021 / acssynbio.0c00140. Available from:47. Liyanagedera SBW, Williams J, Wheatley JP, Biketova AY, Hasan M, Sagona AP, Purdy KJ, Puxty RJ, Feher T, Kulkarni V. SpyPhage: A Cell-Free TXTL Platform for Rapid Engineering of Targeted Phage Therapies. ACS Synth Biol.2022;11(10):3330-3342. Epub 2022 / 10 / 05. doi: 10.1021 / acssynbio.2c00244. Available from:48. Garamella J, Marshall R, Rustad M, Noireaux V. The All E. coli TX-TL Toolbox 2.0: A Platform for Cell-Free Synthetic Biology. ACS Synth Biol.2016;5(4):344-355. Epub 2016 / 01 / 29. doi: 10.1021 / acssynbio.5b00296. Available from:49. Shin J, Jardine P, Noireaux V. Genome replication, synthesis, and assembly of the bacteriophage T7 in a single cell-free reaction. ACS Synth Biol.2012;1(9):408-413. Epub 2013 / 05 / 09. doi: 10.1021 / sb300049p. Available from:50. Rustad M, Eastlund A, Jardine P, Noireaux V. Cell-free TXTL synthesis of infectious bacteriophage T4 in a single test tube reaction. Synth Biol (Oxf).2018;3(1):ysy002. Epub 2018 / 01 / 22. doi: 10.1093 / synbio / ysy002. Available from:51. Vogele K, Falgenhauer E, von Schonberg S, Simmel FC, Pirzer T. Small Antisense DNA-Based Gene Silencing Enables Cell-Free Bacteriophage Manipulation and Genome Replication. ACS Synth Biol.2021;10(3):459-465. Epub 2021 / 02 / 13. doi: 10.1021 / acssynbio.0c00402. Available from:52. Liang J, Zhang H, Tan YL, Zhao H, Ang EL. Directed Evolution of Replication- Competent Double-Stranded DNA Bacteriophage toward New Host Specificity. ACS Synth Biol.2022;11(2):634-643. Epub 2022 / 01 / 29. doi: 10.1021 / acssynbio.1c00319. Available from:43 4859-9106-8900.1

Claims

Atty. Dkt. No.: 136669-0119 CLAIMS 1. A modified bacterial cell lysate composition comprising a mixture of an E. coli lysate and at least one additional bacterial cell lysate obtained from a bacterial host species that is not E. coli, wherein the cell lysate composition comprises an effective amount of transcription / translation (TXTL) machinery that is configured to synthesize at least one bacteriophage that does not target E. coli under cell-free conditions.

2. The modified bacterial cell lysate composition of claim 1, wherein the at least one additional bacterial cell lysate is obtained from a bacterial host species selected from among Bacillus species, Klebsiella species, Pseudomonas species, Acinobacter species, and Staphylococcus species.

3. The modified bacterial cell lysate composition of claim 1 or 2, wherein the E. coli lysate comprises about 1%-99% of the mixture.

4. The modified bacterial cell lysate composition of any of claims 1-3, wherein the at least one additional bacterial cell lysate comprises about 1%-99% of the mixture.

5. A modified bacterial cell lysate composition comprising a mixture of an E. coli lysate and genomic DNA derived from a bacterial host species that is not E. coli, wherein the cell lysate composition comprises an effective amount of transcription / translation (TXTL) machinery that is configured to synthesize at least one bacteriophage that does not target E. coli under cell-free conditions.

6. The modified bacterial cell lysate composition of claim 5, wherein the bacterial host species is selected from among Bacillus species, Klebsiella species, Pseudomonas species, Acinobacter species, and Staphylococcus species.

7. The modified bacterial cell lysate composition of any one of claims 1-6, wherein the composition is prepared using one or more of French-press cell lysis, sonication, runoff reactions, or lysate dialysis.

8. An in vitro method for synthesizing bacteriophage virions comprising a. contacting a bacteriophage genome with the modified bacterial cell lysate composition of any one of claims 1-7, and an energy buffer in vitro to obtain a reaction mixture, and b. incubating the reaction mixture under conditions to produce viable phage virions, wherein the energy buffer comprises canonical amino acids, phosphoenol pyruvate (PEP), nucleoside triphosphates (NTPs), cofactors, and coenzymes. 44 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 9. The method of claim 8, wherein the energy buffer further comprises one or more of a polyethylene glycol (PEG) polymer, deoxynucleotide triphosphates (dNTPs), and stabilizers.

10. The method of claim 9, wherein the PEG polymer comprises PEG-8000 or PEG- 6000.

11. The method of any one of claims 8-10, wherein the energy buffer further comprises one or more of Mg-glutamate, K-glutamate, tRNA, cAMP, folinic acid, spermidine, 3-PGA, HEPES, Nicotinamide adenine dinucleotide (NAD), coenzyme A (CoA), DTT, and maltodextrin.

12. The method of any one of claims 8-11, wherein the reaction mixture further comprises a vector including a nucleic acid sequence encoding a reporter gene, wherein the nucleic acid sequence encoding the reporter gene is operably linked to a promoter that is responsive to a RNA polymerase (RNAP) encoded by the bacteriophage genome.

13. The method of claim 12, wherein the RNAP is selected from among SP6 RNA Polymerase, T3 RNA Polymerase and T7 RNA polymerase.

14. The method of claim 12 or 13, wherein the reporter gene encodes a bioluminescent protein, a fluorescent protein, or a chemiluminescent protein.

15. The method of any one of claims 8-14, wherein the bacteriophage genome is isolated from a naturally occurring bacteriophage, or a genetically engineered bacteriophage or is a synthetic bacteriophage genome.

16. A method for converting a lysogenic bacteriophage into a lytic bacteriophage comprising: generating a plurality of PCR fragments from a DNA template comprising a prophage DNA genome using polymerase chain reaction (PCR), wherein the plurality of PCR fragments collectively span the entire length of the prophage DNA genome except for a gene region encoding integrase; and recombining in vitro the plurality of PCR fragments with a donor heterologous nucleic acid comprising a 5’ flanking region and a 3’ flanking region in the presence of a recombination system under conditions to produce a recombinant lytic bacteriophage genome, wherein at least one end of each PCR fragment comprises a sequence that is homologous to (i) an opposite end of another PCR fragment or (ii) the 5’ flanking region of the donor 45 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 heterologous nucleic acid or (iii) the 3’ flanking region of the donor heterologous nucleic acid.

17. The method of claim 16, wherein the donor heterologous nucleic acid replaces the gene region encoding integrase in the recombinant lytic bacteriophage genome.

18. The method of claim 16 or 17, further comprising propagating the recombinant lytic bacteriophage genome in a bacterial host.

19. The method of claim 16 or 17, further comprising contacting the recombinant lytic bacteriophage genome with a bacterial cell lysate, and an energy buffer in vitro to obtain a reaction mixture, and incubating the reaction mixture under conditions to produce viable phage virions, wherein the energy buffer comprises canonical amino acids, phosphoenol pyruvate (PEP), nucleoside triphosphates (NTPs), cofactors, and coenzymes and wherein the cell lysate comprises an effective amount of transcription / translation (TXTL) machinery that is configured to synthesize bacteriophage under cell-free conditions.

20. A method for generating a genetically modified bacteriophage species comprising generating a plurality of PCR fragments from a DNA template comprising a bacteriophage DNA genome using polymerase chain reaction (PCR), wherein the plurality of PCR fragments collectively span the entire length of the bacteriophage DNA genome except for a first gene region; and recombining in vitro the plurality of PCR fragments with a donor heterologous nucleic acid comprising a 5’ flanking region and a 3’ flanking region in the presence of a recombination system under conditions to produce a genetically modified bacteriophage genome, wherein at least one end of each PCR fragment comprises a sequence that is homologous to (i) an opposite end of another PCR fragment or (ii) the 5’ flanking region of the donor heterologous nucleic acid or (iii) the 3’ flanking region of the donor heterologous nucleic acid, and wherein the donor heterologous nucleic acid replaces the first gene region in the genetically modified bacteriophage genome.

21. The method of claim 20, further comprising propagating the genetically modified bacteriophage genome in a bacterial host.

22. The method of claim 20, further comprising 46 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 contacting the genetically modified bacteriophage genome with a bacterial cell lysate, and an energy buffer in vitro to obtain a reaction mixture, and incubating the reaction mixture under conditions to produce viable phage virions, wherein the energy buffer comprises canonical amino acids, phosphoenol pyruvate (PEP), nucleoside triphosphates (NTPs), cofactors, and coenzymes and wherein the cell lysate comprises an effective amount of transcription / translation (TXTL) machinery that is configured to synthesize bacteriophage under cell-free conditions.

23. The method of claim 19 or 22, wherein the bacterial cell lysate is obtained from a naturally occurring bacterial host cell or a genetically modified bacterial host cell.

24. The method of claim 23, wherein the genetically modified bacterial host cell overexpresses one or more of translation initiation factor IF-3 (infC), OxyS and CyaR and / or represses RecC subunit exonuclease RecBCD.

25. The method of claim 19 or 22, wherein the bacterial cell lysate comprises the modified bacterial cell lysate composition of any one of claims 1-7.

26. The method of any one of claims 19 or 22-25, wherein the energy buffer further comprises one or more of a polyethylene glycol (PEG) polymer, deoxynucleotide triphosphates (dNTPs), and stabilizers.

27. The method of claim 26, wherein the PEG polymer comprises PEG-8000 or PEG- 6000.

28. The method of any one of claims 19 or 22-27, wherein the energy buffer further comprises one or more of Mg-glutamate, K-glutamate, tRNA, cAMP, folinic acid, spermidine, 3-PGA, HEPES, Nicotinamide adenine dinucleotide (NAD), coenzyme A (CoA), DTT, and maltodextrin.

29. The method of any one of claims 19 or 22-28, wherein the reaction mixture further comprises a vector including a nucleic acid sequence encoding a reporter gene, wherein the nucleic acid sequence encoding the reporter gene is operably linked to a promoter that is responsive to a RNA polymerase (RNAP) encoded by the bacteriophage genome.

30. The method of claim 29, wherein the RNAP is selected from among SP6 RNA Polymerase, T3 RNA Polymerase and T7 RNA polymerase.

31. The method of claim 29 or 30, wherein the reporter gene encodes a bioluminescent protein, a fluorescent protein, or a chemiluminescent protein. 47 4859-9106-8900.1Atty. Dkt. No.: 136669-0119 32. The method of any one of claims 16-31, wherein the recombination system comprises a 5’-3’ exonuclease, a DNA polymerase, and a DNA ligase.

33. The method of any one of claims 16-32, wherein the donor heterologous nucleic acid comprises an open reading frame that encodes a bioluminescent protein, a fluorescent protein, a chemiluminescent protein, a heterologous bactericidal gene, a phage protein that modifies host range, or any combination thereof.

34. The method of claim 33, wherein the open reading frame of the donor heterologous nucleic acid is operably linked to an expression control sequence that is capable of directing expression of the bioluminescent protein, the fluorescent protein, the chemiluminescent protein, the heterologous bactericidal gene, a phage protein that modifies host range, or any combination thereof, optionally wherein the expression control sequence is an inducible promoter or a constitutive promoter.

35. The method of claim 14, 31, 33, or 34, wherein the bioluminescent protein is Aequorin, firefly luciferase, Renilla luciferase, red luciferase, luxAB, or nanoluciferase.

36. The method of claim 14, 31, 33, or 34, wherein the chemiluminescent protein is β- galactosidase, horseradish peroxidase (HRP), or alkaline phosphatase.

37. The method of claim 14, 31, 33, or 34, wherein the fluorescent protein is TagBFP, Azurite, EBFP2, mKalama1, Sirius, Sapphire, T-Sapphire, ECFP, Cerulean, SCFP3A, mTurquoise, monomeric Midoriishi-Cyan, TagCFP, mTFP1, EGFP, Emerald, Superfolder GFP, Monomeric Azami Green, TagGFP2, mUKG, mWasabi, EYFP, Citrine, Venus, SYFP2, TagYFP, Monomeric Kusabira-Orange, mKOκ, mKO2, mOrange, mOrange2, mRaspberry, mCherry, dsRed, mStrawberry, mTangerine, tdTomato, TagRFP, TagRFP-T, mApple, mRuby, mPlum, HcRed-Tandem, mKate2, mNeptune, NirFP, TagRFP657, IFP1.4, iRFP, mKeima Red, LSS-mKate1, LSS- mKate2, PA-GFP, PAmCherry1, PATagRFP, Kaede (green), Kaede (red), KikGR1 (green), KikGR1 (red), PS-CFP2, PS-CFP2, mEos2 (green), mEos2 (red), PSmOrange, or Dronpa.

38. The method of any one of claims 20-37, wherein the genetically modified bacteriophage species is genetically modified T7 phage. 48 4859-9106-8900.1

Images