37 results about "Cell-free system" patented technology

The application provides an in-vitro cell-free protein synthesis system and kit containing exogenous magnesium ions and application thereof, including a D2P system (DNA-to-Protein system) and an mR2P system (mRNA-to-Protein system), and belongs to the technical field of protein synthesis. The in-vitro cell-free protein synthesis system containing exogenous magnesium ions adopts magnesium aspartate as a novel magnesium ion source, and especially in a eukaryotic cell-free system, compared with a traditional magnesium ion source, can significantly improve the protein synthesis efficiency and protein expression amount. A more efficient and higher-throughput in-vitro protein synthesis kit and a synthesis method of exogenous proteins are also provided, and the kit has the advantages of simplicity, convenience and low cost.

According to the application of the controllable delivery of the multi-membraneless compartment interlamellar substances in the cell-free system, two independent and coexisting protein enrichment phases are rapidly formed in the optimized cell-free system by utilizing the protein expression of a protein expression vector and adding histidine tag (His Tag) protein, namely, after the multi-membraneless compartment is formed, the two independent and coexisting protein enrichment phases are separated from the cell-free system, and the cell-free system can be used for controlling the delivery of the multi-membraneless compartment interlamellar substances in the cell-free system. And controllable delivery of micromolecular substances and macromolecular substances in multiple membrane-free zone chambers is carried out. The multiple membrane-free compartments constructed by the invention can controllably enrich specific active components and biochemical reactions in a cell-free system into the compartments and separate the specific active components and biochemical reactions from other complex metabolic reactions, and due to the high dynamic nature of the compartments, controllable communication between biomolecules and the external environment and among multiple compartments can be realized; further, the accuracy and controllability of metabolic reaction are greatly improved, and a solid foundation is laid for application of a cell-free system in the aspects of complex metabolic pathway exploration, multi-enzyme catalysis, drug delivery and the like.

Provided is a method with which it is possible to simply and exponentially amplify circular DNA, particularly long-chain circular DNA, in a cell-free system. Specifically, provided is a method for amplifying circular DNA, which comprises an isothermal reaction of a reaction mixture produced by mixing circular DNA having a replication initiation sequence (oriC) with a reaction solution containing the following enzyme groups: (1) a first enzyme group that catalyzes replication of the circular DNA, (2) a second enzyme group that catalyzes replication of the circular DNA, (3) a third enzyme group that catalyzes replication of the circular DNA, (4) a fourth enzyme group that catalyzes replication of the circular DNA, and (5) a third enzyme group that catalyzes replication of the circular DNA. (2) a second enzyme group which catalyzes a linking reaction of an okazaki fragment to synthesize two sister circular DNAs forming a catenary body; and (3) a third enzyme group that catalyzes a separation reaction of the two sister circular DNAs.

Provided is a method capable of replicating or amplifying circular DNA, and particularly, long-chain circular DNA, in a cell-free system. Specifically, provided is a method for suppressing generation of a DNA multimer as a by-product, when circular DNA having a replication origin sequence (origin of chromosome (oriC)) is replicated or amplified by using the following enzyme groups: (1) a first enzyme group that catalyzes replication of circular DNA; (2) a second enzyme group that catalyzes an Okazaki fragment maturation and synthesizes two sister circular DNAs constituting a catenane; and (3) a third enzyme group that catalyzes a separation of two sister circular DNAs. Moreover, also provided is a method comprising introducing oriC into circular DNA by using a transposon.

The invention relates to a method for constructing a membrane-free compartment in a cell-free system containing exogenous nickel ions, which comprises the following steps of: constructing a protein expression vector containing a histidine tag (His Tag), introducing the protein expression vector into an expression host, and adding protein obtained by expression and purification into the cell-free system containing the exogenous nickel ions to directly form a protein enrichment phase, namely the membrane-free compartment. The biosynthesis of the quantum dots can be promoted in the zone chamber, the biological enzyme with the histidine tag can be recruited in a high-specificity manner, the enzyme digestion reaction is completed in the zone chamber, and the substances are controllably released.

The present disclosure relates to multi-enzyme pathways for the in vitro cell-free biosynthesis of the cofactor, coenzyme A ("CoA") from lower-cost substrates, such as D-pantothenate, L-cysteine, and ATP, and the use of these pathways in CoA-dependent cell-free systems for the biobased production of chemicals, such as acetyl-CoA, hexanoyl-CoA, malonyl-CoA, and cannabigerolic acid (CBGA).

Compositions and methods disclosed herein relate to improved in vitro cell-free systems for various biological reactions. In one aspect, a composition of the present disclosure includes: a cell-free extract derived from an organism; a nucleic acid; and an organelle that is heterologous to the organism.

The present invention provides: a method for easily isolating a plurality of gene species from the gene species in a cell-free system; and a method for easily isolating and selecting a specific gene species in a cell-free system using indexes derived from individual gene species. The present invention is a DNA amplification method comprising: preparing a mixture of DNA that is circular DNA having a replication origin sequence capable of binding to an enzyme having a DnaA activity; preparing a reaction gel solution containing the mixture, a first enzyme group which catalyze the replication of the circular DNA, a second enzyme group which synthesize two sister circular DNA molecules that catalyze an Okazaki fragment linking reaction to form a catenane, a third enzyme group which catalyze the reaction for separating the two sister circular DNA molecules, and a gelatinizing agent; gelatinizing the reaction gel solution to produce an amplification gel plate containing the circular DNA; and subjecting the DNA to a circular DNA amplification reaction at a temperature of 80°C or lower using the gel plate to produce amplified DNA.

The disclosure provides biosynthetic platforms that generate olivetolic acid and its analogues at high titers from microbes, and in cell free systems.

This disclosure relates to single-molecule tracking methods, including high-throughput methods, for analyzing molecules in cell-free samples. The disclosure further provides a system for carrying out such methods.

Provided herein are membrane encapsulated cell-free systems comprising transcriptionally regulated biosensors for detecting analytes, and methods of their use. In embodiments, the biosensor is a riboswitch for detection of fluoride in a sample.

The application discloses a paper-based arsenic ion synthetic biosensor based on a cell-free system and application thereof, and comprises a cell-free arsenic ion sensing module and a semi-quantitative paper-based detection chip. The cell-free arsenic ion sensing module is composed of a synthetic arsenate reduction module, an arsenic ion specificity sensing module, a high-gain transcription amplification module and a visual reporting module connected in sequence. The synthetic arsenate reduction module converts pentavalent arsenic into trivalent arsenic; the arsenic ion specificity sensing module recognizes trivalent arsenic and removes transcription inhibition; the high-gain transcription amplification module amplifies the signal through ecf11 cascade; and the visual reporting module outputs a colorimetric or fluorescent signal. The application integrates an engineered promoter and an ecf11 cascade amplification system in a cell-free system, greatly improves sensitivity and dynamic range and keeps low leakage. The paper-based chip can present different pattern arrangements according to arsenic concentration and is easy to read. The application provides an environmentally friendly, portable and easy-to-read innovative method for monitoring arsenic pollution in drinking water.

Provided is an enzyme useful for prenylation and recombinant pathways for the production of cannabinoids, cannabinoid precursors and other prenylated chemicals in a cell free system as well and recombinant microorganisms that catalyze the reactions.

The present disclosure relates to single molecule tracking methods, including high throughput methods, for analyzing molecules in cell-free samples. The present disclosure further provides systems for performing such methods.

A method for constructing multiple membraneless compartments in a cell-free system is characterized in that at least two membraneless compartments are constructed by adding phase separation protein into the cell-free system or by means of biosynthesis of the cell-free system. The membrane-free zone chamber constructed by the invention can controllably enrich specific active components and biochemical reactions in a cell-free system into the zone chamber and separate the specific active components and biochemical reactions from other complex metabolic reactions. Due to the high dynamic nature of the compartments, cooperation of the compartments can be achieved through the multiple membrane-free compartments, and regulation and control of the collection amount of compartment products are jointly completed.

The present invention relates to a cell lysate wherein the lysis buffer comprises magnesium acetate, the phosphorylation inhibitor K3L, the agonist of dephosphorylation GADD34 and a caspase inhibitor for use in, or as, a cell-free system and a method for preparing a cell-free protein synthesis system. Further provided is a method for expressing a protein in the cell-free protein synthesis system, a method for screening for a candidate compound for an effect on a target molecule and a use of the cell-free protein synthesis system. The present invention also provides a kit for the above-mentioned cell-free systems.

Provided herein are methods and cell-free systems for producing recombinant antibodies, wherein the antibodies are directed against target proteins naturally expressed as intracellular proteins. Also provided herein are modified cell-free systems for use in such methods, wherein the modified system is engineered to express a variant target protein having a mutant epitope of a target epitope of an antibody.

The invention relates to a method for preparing scDNA, which comprises the following steps: providing a circular nucleic acid template which is double-stranded and comprises a to-be-recombined ring-forming region and a to-be-enzyme-digested incision site arranged outside the region, and the to-be-recombined ring-forming region comprises an expression framework and a recombination site pair arranged on two wings of the expression framework; the present invention relates to a method for preparing a cyclic nucleic acid template, the cyclic nucleic acid template being amplified by RCA rolling circle amplification to form a concatemer, the concatemer comprising a tandem repeat unit of a cyclic nucleic acid template sequence, the concatemer being linearized by enzyme digestion in the incision site of the tandem repeat unit with a restriction enzyme simultaneously with or subsequent to the rolling circle amplification, the linearized concatemer comprising a cyclic nucleic acid template sequence, the cyclic nucleic acid template comprising a cyclic nucleic acid template sequence, and the cyclic nucleic acid template sequence comprising a cyclic nucleic acid template sequence. Free linearized nucleic acid is obtained; in a cell-free system, the linearized nucleic acid is recombined with a recombinase into scDNA comprising the expression framework. The method provided by the invention really realizes efficient preparation of scDNA.

The invention provides a cell-free toxin synthesis system and method. The system at least comprises: (1) a cell-free system; (2) an amplification system containing amplified plasmids of the cell-free system is added, the plasmids are plasmids containing coding toxin protein, the amplification system containing the amplified plasmids is obtained after the plasmids are added into the amplification system according to the preset concentration to be amplified, the preset concentration is 2-10 ng / mu L, and the preset concentration is not 2 ng / mu L. According to the method, plasmids with a certain concentration are added into a cell-free reaction system, so that the in-vitro expression efficiency of toxins is effectively improved, and particularly, the purification efficiency is more obviously improved.

The invention relates to cell-free systems, methods, and kits for bio-manufacturing natural or chemical products from readily available feedstocks, such as glucose. The systems, methods, and kits allow for cell-free bio-manufacturing of desired products in cell-free conditions, and the rapid optimization of conditions for preparing the products in cell-free conditions. Disclosed herein are systems, methods, and kits for the cell-free production of fatty acids, cannabinoids, and their intermediates.

Provided herein are methods and cell-free systems for producing recombinant antibodies, wherein the antibodies are directed against target proteins naturally expressed as intracellular proteins. Also provided herein are modified cell-free systems for use in such methods, wherein a cell-free protein expression system is modified by an agent that binds to an intracellular target protein or antibody complimentary and thus prevents binding of an antibody to a target protein.

Systems and methods for the production of isopentenylation and recombinant pathways of compounds, cannabinoids, cannabinoid precursors, and other isopentenylation chemicals in cell-free systems. The invention also includes enzymes and recombinant microorganisms that catalyze the reaction. The compounds, cannabinoids, cannabinoid precursors and other isopentenylation chemicals can be substituted with at least one deuterium, at least one tritium, at least one halogen, at least one hydroxyl group and / or at least one additional isotope.

The present disclosure relates to multi-enzyme pathways for the in-situ generation of the cofactor NADP from cost-effective reagents such as nicotinate, NMN (nicotinamide mononucleotide) and NAD (nicotinamide adenine dinucleotide) and the use of these pathways in NADP-dependent cell-free systems for the bio-based production of chemicals such as isobutanol.

The present invention relates to preparation of key drug intermediate, L-2-amino butyric acid (L-2-ABA) by a method of cell free system and biotransformation using genetically engineered strains from easily available economic substrates like citramalate or citraconate and enzymes like LeuCD, LeuB and ValDH or IlvE.

The present disclosure relates to single molecule tracking methods, including high-throughput methods, for analyzing molecules in cell-free samples. The present disclosure also provides systems for performing such methods.

The invention discloses a preparation method of tomato leaf vacuole-free cytoplasm. The method comprises the following steps: cutting tomato leaves, putting the cut tomato leaves into an enzymolysis buffer solution containing cellulase, macerozyme, mannitol, potassium chloride, MES, calcium chloride, beta-mercaptoethanol and fetal calf serum, and carrying out vacuumizing enzymolysis to obtain protoplast; removing vacuoles through density gradient centrifugation, and collecting tiny protoplasts; and performing staged homogenate cracking and centrifugal purification to obtain the tomato leaf vacuole-free cytoplasm. The vacuole-free cytoplasm prepared by the invention has dsRNA cleavage activity, and can be used for tomato virus in-vitro research and plant cell-free system research and development.

Provided is a method capable of simply and exponentially amplifying circular DNA, and particularly, long-chain circular DNA, in a cell-free system. Specifically, provided herein is a method for amplifying circular DNA which comprises mixing circular DNA having a replication origin sequence (origin of chromosome (oriC)) with a reaction solution comprising: a first enzyme group that catalyzes replication of circular DNA; a second enzyme group that catalyzes an Okazaki fragment maturation and synthesizes two sister circular DNAs constituting a catenane; a third enzyme group that catalyzes a separation of two sister circular DNAs; and also, a buffer, NTP, dNTP, a magnesium ion source, and an alkali metal ion source, to form a reaction mixture, which is then reacted.

Described are methods for generating a reporter molecule in response to a target analyte in a cell-free system. A synthetic biological circuit is used to modify the level of the reporter molecule in response to the presence of the target analyte. The reporter molecule may be glucose or another molecule readily detected using a device such as glucose monitor or other portable sensor. Also provided are kits comprising a cell-free system with a synthetic biological circuit that generates or consumes a reporter molecule in response to a target analyte.

The invention belongs to the technical field of biomedicine, and particularly relates to a KV1.3 expression vector, a cell-free expression system, a recombination channel and application thereof. The recombinant expression vector is obtained by inserting a DNA (deoxyribonucleic acid) sequence as shown in SEQ ID NO.1 between an Nde I site and an Xho I site of a pIVEX-2. 4d vector, and the recombinant expression vector is named as pIVEX-K. The cell-free expression system is obtained by transferring the expression vector into a cell-free system. The recombination channel is obtained by recombining a cell-free expression system product and lipidosome. The recombinant channel can be applied to high-throughput screening of compounds influencing the potassium ion channel function, the influence of the compounds on the potassium channel function is directly displayed by monitoring the change of a fluorescence signal before and after a to-be-detected object is added, and the recombinant channel has the characteristics of simplicity in operation, short test period and high sensitivity.