22 results about "Superhelix" patented technology

The invention provides a production strain of an antibiotic-free plasmid, the production strain is a gene editing strain of a PIR strain and is named as PIR1-WN:: 0636 or PIR1-PR: 0636, the production strain contains a nucleotide sequence for coding toxin protein and the antibiotic-free plasmid, and the antibiotic-free plasmid contains a nucleotide sequence for coding antitoxin protein; and preferably, the replicon DNA element of the nonreactive plasmid is R6K-gamma. The toxin protein gene of the production strain disclosed by the invention can be stably passaged, has lethality after being induced and can be used for plasmid screening; according to the invention, the positive rate of transforming the nonreactive plasmid into the PIR1-WN:: 0636 strain is more than 80%, and stable production of the plasmid with a high superhelix ratio can be realized.

Methods of assessing double-stranded plasmid DNA isoforms to determine a proportion of supercoiled plasmid DNA compared to linear plasmid DNA, and / or open circle plasmid DNA are provided according to aspects of the present disclosure, which include: preferentially labelling double-stranded plasmid DNA; flowing the labeled double-stranded plasmid DNA through a polymeric separation medium in a microchannel into a detection region; detecting the detectable label to determine: a) an amount of time taken by the labeled plasmid DNA isoforms to flow through the polymeric separation medium in the microchannel into the detection region, and / or b) strength of the signal of the detectable label in the detection region; and comparing a) and / or b) to a reference standard thereby determining a proportion of supercoiled plasmid DNA compared to linear plasmid DNA, and / or open circle plasmid DNA in the fluid sample comprising plasmid DNA.

Kits for assessing a proportion of supercoiled plasmid DNA compared to linear plasmid DNA, and / or a contaminant, in a fluid sample containing plasmid DNA according to aspects of the present disclosure include: a DNA reference standard including supercoiled pDNA and / or linear pDNA. Methods of assessing double-stranded plasmid DNA in a fluid sample according to aspects of the present disclosure include comparing characteristics of double-stranded plasmid DNA in a fluid sample to a reference standard representative of one or both of: supercoiled plasmid DNA and linear plasmid DNA, wherein the reference standard includes 1) at least two fragments of supercoiled pDNA of specified sizes and / or 2) at least two fragments of linear pDNA of specified sizes.

The invention relates to a method for early-stage removal of endotoxin in plasmid DNA preparation. The method comprises the following steps: (1) carrying out microfiltration on a lysis neutralization solution to obtain a filtrate; (2) concentrating the filtrate to obtain a concentrated solution, exchanging the obtained concentrated solution with purified water or a 10 * TE buffer solution, and adjusting the pH value to 4.4-5.6 with acetic acid to obtain an acidic solution; (3) adding zinc sulfate with the final concentration of 0.5-1M into the acidic solution, and incubating at 10-20 DEG C for 20-60 minutes to obtain an incubation solution; and (4) carrying out microfiltration on the incubation liquid, and carrying out ultrafiltration replacement on the obtained filtrate to remove the precipitate. The early clearing method for endotoxin in plasmid DNA preparation is efficient, economical and safe, endotoxin pollution can be controlled from the source, the proportion and yield of superhelix plasmids are considered, and the quality of plasmid DNA is improved. The early clearing method for endotoxin in plasmid DNA preparation can simplify the downstream purification process, and is especially suitable for large-scale industrial production of high-quality plasmid DNA.

The invention discloses a recombinant topoisomerase and an application of the recombinant topoisomerase in plasmid linearization. The invention discloses a recombinant topoisomerase with an amino acid sequence as shown in SEQ ID NO.1 at first. The invention further discloses an application of the recombinant topoisomerase in plasmid linearization. The recombinant topoisomerase does not depend on recognition of a specific DNA sequence, cutting sites can be formed on plasmids in a super-spiral form, then a linear product is obtained through the cutting effect of T7 endonuclease I, and linearization of plasmids with unknown sequences can be achieved. The plasmid linearization kit developed by using the recombinant topoisomerase has the characteristics of rapidness, high efficiency and wide applicability, and can realize full-length sequencing of unknown plasmids by combining with a three-generation sequencing technology.

Methods of assessing double-stranded plasmid DNA isoforms to determine a proportion of supercoiled plasmid DNA compared to linear plasmid DNA, and / or open circle plasmid DNA are provided according to aspects of the present disclosure, which include: preferentially labelling double-stranded plasmid DNA; flowing the labeled double-stranded plasmid DNA through a polymeric separation medium in a microchannel into a detection region; detecting the detectable label to determine: a) an amount of time taken by the labeled plasmid DNA isoforms to flow through the polymeric separation medium in the microchannel into the detection region, and / or b) strength of the signal of the detectable label in the detection region; and comparing a) and / or b) to a reference standard thereby determining a proportion of supercoiled plasmid DNA compared to linear plasmid DNA, and / or open circle plasmid DNA in the fluid sample comprising plasmid DNA.

The invention discloses a quantitative detection method for plasmid residues after cleaning of superhelical plasmid collinear production equipment, which comprises the following steps: (1) confirming the configuration of residual plasmids according to the electrophoresis result of a cleaning solution after half cleaning, selecting plasmid standard substances according to the configuration of the residual plasmids, preparing standard substance solutions and leacheate with different concentrations, and carrying out ultrasonic treatment on the standard substance solutions and the leacheate; the method comprises the following steps: (1) preparing a qPCR reaction system, (2) preparing a qPCR reaction system which comprises a qPCR special premix solution, a forward primer, a reverse primer, a 5'end labeled reporter group, a 3 'end labeled quenching group specific probe, a 50 * ROX reference dye II, a standard solution or eluent and a diluent, (3) setting a qPCR reaction program to carry out an amplification reaction, and (4) drawing a standard curve and calculating the residual quantity of plasmids. The detection method provided by the invention not only has a very low detection limit and can avoid low systematicness of a measurement result, but also is more accurate in detection result and can avoid high result falseness, so that accurate and reliable cleaning verification is realized.

The invention discloses a method for producing mRNA (messenger ribonucleic acid) template plasmids by fermenting escherichia coli, which comprises the following steps: firstly, inoculating a recombinant escherichia coli seed solution into a basic culture medium, fermenting and culturing until the OD600 value is 18-22, and then continuously adding a fed-batch culture medium at the feeding rate of 11-23 mL / L / h, the seed culture medium contains 1-5 mmoL / L of metal ion chelating agent and 0.5-1.5 g / L of anhydrous magnesium sulfate, the carbon source of the basic culture medium is 15-25 g / L of glycerol, and the pH value of the basic culture medium is 7-8. The culture medium contains 3-21 mmoL / L of a metal ion chelating agent and 1.5-4.5 g / L of anhydrous magnesium sulfate, the carbon source of the fed-batch culture medium is 260-450 g / L of glycerol, and the metal ion chelating agent is preferably sodium citrate. The method optimizes and reduces the aggregate superhelix content of the mRNA template plasmid from the aspects of culture medium formula and culture condition, and is beneficial to large-scale production of high-quality target mRNA template plasmid.

Current minicircle production methods are slow, expensive, and difficult to perform under GMP conditions because, in most cases, the product is derived from bacteria. In contrast, HTLA-and CHTLA-based synthetic circular supercoiled DNA production can be done completely in a test tube, using chemically or enzymatically synthesized oligonucleotides, long single stranded DNA, and / or double stranded DNA.

Kits for assessing a proportion of supercoiled plasmid DNA compared to linear plasmid DNA, and / or a contaminant, in a fluid sample containing plasmid DNA according to aspects of the present disclosure include: a DNA reference standard including supercoiled pDNA and / or linear pDNA. Methods of assessing double-stranded plasmid DNA in a fluid sample according to aspects of the present disclosure include comparing characteristics of double-stranded plasmid DNA in a fluid sample to a reference standard representative of one or both of: supercoiled plasmid DNA and linear plasmid DNA, wherein the reference standard includes 1) at least two fragments of supercoiled pDNA of specified sizes and / or 2) at least two fragments of linear pDNA of specified sizes.

The invention relates to a method for purifying superhelical plasmid DNA (deoxyribonucleic acid). The method comprises the following steps: (1) performing microfiltration treatment on a thallus lysis solution containing target plasmids to obtain filtrate; (2) concentrating the filtrate through a hollow fiber column by using a tangential flow filtration system to obtain a concentrated solution; and (3) changing the concentrated solution through a hollow fiber column by using a tangential flow filtration system, wherein the adopted replacement solution is a 80-120mM Tris-HCl buffer solution containing 8-12mM EDTA (Ethylene Diamine Tetraacetic Acid). According to the method disclosed by the invention, the super-spiral plasmid purification is carried out through tangential flow filtration concentration and liquid exchange operation for the first time, a plasmid skeleton and a target gene sequence do not need to be optimized, chromatographic treatment is also not needed, the aggregate proportion can be greatly reduced while the plasmid DNA yield is ensured, and the super-spiral DNA purity is improved. The method disclosed by the invention is simple and rapid to operate, good in applicability and suitable for large-scale production of high-quality and high-purity superhelix plasmid DNA.

Methods of assessing double-stranded plasmid DNA isoforms to determine a proportion of supercoiled plasmid DNA compared to linear plasmid DNA, and / or open circle plasmid DNA are provided according to aspects of the present disclosure, which include: preferentially labelling double-stranded plasmid DNA; flowing the labeled double-stranded plasmid DNA through a polymeric separation medium in a microchannel into a detection region; detecting the detectable label to determine: a) an amount of time taken by the labeled plasmid DNA isoforms to flow through the polymeric separation medium in the microchannel into the detection region, and / or b) strength of the signal of the detectable label in the detection region; and comparing a) and / or b) to a reference standard thereby determining a proportion of supercoiled plasmid DNA compared to linear plasmid DNA, and / or open circle plasmid DNA in the fluid sample comprising plasmid DNA.

The invention relates to the technical field of biology, in particular to a plasmid DNA purification method based on membrane chromatography. According to the method, a membrane chromatography technology is used, a packing experiment is not needed, the working flow speed is high, plasmid purification only needs 1-2 h once, the recovery rate of obtained DNA is 73% or above, the recovery rate of super-spiral DNA is 97% or above, and the proportion of the super-spiral DNA is 95% or above.

The subject invention provides fluorophore-quencher nucleic acid molecules comprising relaxed or supercoiled DNA molecules, and their use in rapid and efficient high-throughput screening (HTS) assays to screen and identify compounds that inhibit DNA gyrases. These compounds can be used as antibiotics for treating bacterial infections, especially, multidrug resistant bacterial infections.

Present methods for the production of microrings are slow, expensive and difficult to carry out under GMP conditions, because in most cases, the product is derived from bacteria. In contrast, synthetic circular superhelix DNA production based on HTLA and CHTLA can be completed completely in a test tube, using chemically or enzymatically synthesized oligonucleotides, long single-stranded DNA, and / or double-stranded DNA.

This invention relates to a method for controlling the cleavage of DNA products by low-dimensional nanomaterials using an applied electric field. The method includes constructing a microdroplet three-electrode electrochemical system. This system comprises a working electrode containing low-dimensional nanomaterials, an electrolyte containing target DNA, a counter electrode, and a reference electrode. The reference electrode provides a potential reference, and the counter electrode forms a current loop with the working electrode. Using the reference electrode as the potential reference, a constant voltage is continuously applied to the working electrode. By controlling the potential of this constant voltage, the conformation of the DNA at the interface between the working electrode and the electrolyte changes, thereby controlling the conformational changes of DNA from supercoiled to open-ring, linear, etc. This invention overcomes the technical bottleneck of the traditional homogeneous reaction system between low-dimensional nanomaterials and DNA, which suffers from single and uncontrollable products. It offers advantages such as mild reaction conditions, high control precision, fast response speed, good product selectivity, reusable catalyst, and strong system compatibility.

The subject invention provides fluorophore-quencher nucleic acid molecules comprising relaxed or supercoiled DNA molecules, and their use in rapid and efficient high-throughput screening (HTS) assays to screen and identify compounds that inhibit DNA gyrases. These compounds can be used as antibiotics for treating bacterial infections, especially, multidrug resistant bacterial infections.

The application provides a method for detecting the purity of supercoiled DNA in plasmid samples with different molecular weights and application thereof, and comprises the following steps: preparing linearized plasmid samples and untreated plasmid samples for HPLC detection; performing HPLC detection by using an anion exchange column; collecting chromatograms of supercoiled plasmid DNA, open circle plasmid DNA, linear plasmid DNA and concatemer plasmid DNA in linearized and untreated plasmid samples with different molecular weights; and the mobile phase of the HPLC detection comprises: mobile phase A is a water solution containing tris-hydroxymethyl aminomethane, and mobile phase B is a water solution containing tris-hydroxymethyl aminomethane and sodium chloride. The method can ensure complete separation of supercoiled plasmid DNA, open circle plasmid DNA, linear plasmid DNA and concatemer plasmid DNA in plasmid samples with different molecular weights, has high resolution and high sensitivity, and has important application value in the field of nucleic acid drug analysis technology.

The application provides a protein, a self-replicating mRNA molecule and a rabies virus saRNA vaccine. The amino acid sequence of the protein is shown as SEQ ID NO: 5, the mRNA molecule comprises coding region RNA of the amino acid sequence, preferably, the nucleotide sequence of the mRNA molecule is shown as SEQ ID NO: 6, the vaccine is a lipid nanoparticle comprising the mRNA molecule shown as SEQ ID NO: 6, and additionally, the application also discloses a preparation method of the rabies virus saRNA vaccine, which comprises the steps of saRNA molecule design, supercoiled plasmid preparation, preparation of the supercoiled plasmid into a linear plasmid template, preparation of a saRNA stock solution, LNP-saRNA preparation and the like. On the basis of scientifically designing a target gene molecule, the rabies virus saRNA vaccine obtained by the application has the advantages of low use dosage, long duration of immune protection, an injection dosage of 5 mu even lower, LNP-saRNA secondary injection immunization, and a duration of immune protection of more than 126 days. The vaccine has good protection effect, and the NIH titer is as high as 217.4 IU / mL or higher, which is much higher than the reference vaccine of 11.4 IU / mL.

Methods of assessing double-stranded plasmid DNA isoforms to determine a proportion of supercoiled plasmid DNA compared to linear plasmid DNA, and / or open circle plasmid DNA are provided according to aspects of the present disclosure, which include: preferentially labelling double-stranded plasmid DNA; flowing the labeled double-stranded plasmid DNA through a polymeric separation medium in a microchannel into a detection region; detecting the detectable label to determine: a) an amount of time taken by the labeled plasmid DNA isoforms to flow through the polymeric separation medium in the microchannel into the detection region, and / or b) strength of the signal of the detectable label in the detection region; and comparing a) and / or b) to a reference standard thereby determining a proportion of supercoiled plasmid DNA compared to linear plasmid DNA, and / or open circle plasmid DNA in the fluid sample comprising plasmid DNA.

The present invention relates to a composition comprising a transposase, optionally a RNA-guided nuclease, and a supercoiled circular dsDNA molecule comprising a transgene flanked by ITRs, in particular for its use as a gene editing tool.

Provided is an antibiotic resistance gene-free plasmid-containing production strain. The production strain is a gene-edited strain of the PIR strain, designated as PIR1-WN::0636 or PIR1-PR::0636. The production strain contains a nucleotide sequence encoding a toxin protein and an antibiotic resistance gene-free plasmid, wherein the antibiotic resistance-free plasmid contains a nucleotide sequence encoding an antitoxin protein; preferably, the replicon DNA element for the antibiotic resistance gene-free plasmid is R6K-γ. The toxin protein gene of the provided production strain can be stably maintained during strain passage, and has lethality upon induction, which can be used for plasmid screening. When the provided antibiotic resistance gene-free plasmid is transformed into the PIR1-WN::0636 strain, the positive rate reaches 80% or higher. Moreover, the antibiotic resistance gene-free plasmid can enable the stable production of plasmids with a high supercoiled proportion.