36 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.

The invention discloses an unmanned mine car slip rate control method based on superhelix second-order sliding mode control. The method comprises the following steps: deriving a second-order slip rate system kinetic equation based on a wheel kinetic model; introducing a steepest differential tracker to carry out slip rate differential estimation; converting the second-order slip rate kinetic equation into an error variable form for expression; designing a non-singular sliding mode surface according to a fixed time convergence theorem, and calculating the control rate of second-order sliding mode control based on a superhelix algorithm: issuing a braking torque control signal to a hydraulic braking control unit of the unmanned mine car, and further adjusting the braking pressure of a wheel cylinder. The method has the advantages that the actual slip rate of the wheels is controlled to be close to the target slip rate, the locking phenomenon is prevented when the mine car is braked, and the braking efficiency and safety of the mine car are improved.

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 utility model relates to the technical field of experimental appliances, and discloses a superhelical DNA separation and purification column test tube rack which comprises a bottom plate, connecting rods are fixedly connected to the four corners of the top of the bottom plate, a placing plate is slidably connected to the outer portions of the connecting rods, and a plurality of connecting frames are fixedly connected to the front side and the rear side of the placing plate. A limiting assembly is fixedly connected to the outer portion of the connecting frame, a rotating plate is rotatably connected to the outer portion of the connecting frame, a first spring is arranged in the placing plate, a clamping rod is fixedly connected to the outer portion of the rotating plate, a plurality of clamping grooves are formed in the outer portion of a connecting rod, and a liquid storage tank is fixedly connected to the bottom of the bottom plate. According to the test tube rack, the height of the test tube rack is adjusted, and the test tube rack can adapt to test tubes with different sizes and heights by adjusting the height, so that the flexibility of an experiment is improved, and various test tubes with different specifications can be used in the same test, so that different experiment requirements are met.

The invention discloses a RuY conjugate of a coumarin modified metal ruthenium derivative and a preparation method and application of the RuY conjugate, metal ruthenium in molecules of the RuY conjugate carries positive charges, and compared with traditional small organic molecules, the RuY conjugate has the advantage that the bacterial cell membrane penetrating capacity and the retention effect of the RuY conjugate are enhanced. Researches find that part of coumarin can be combined with a B subunit of DNA gyrase in bacteria, and DNA superhelix is inhibited by blocking ATP enzyme activity. Therefore, the coumarin derivative has potential antibacterial activity. Tests prove that the RuY conjugate disclosed by the invention can effectively inhibit the growth of aeromonas veronii, nocardia and vancomycin-resistant enterococcus faecium, so that the RuY conjugate is used for preparing medicines for preventing and treating diseases caused by the aeromonas veronii, nocardia and vancomycin-resistant enterococcus faecium, and the RuY conjugate disclosed by the invention provides a new direction for the development of novel antibacterial agents.

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 present disclosure provides a method for isolating supercoiled plasmid DNA (pDNA) from a liquid sample, comprising: The present invention provides a method for separating pDNA from pDNA comprising the steps of: (a) applying a liquid sample comprising pDNA to a first chromatographic material comprising (i) an anion exchange chromatographic ligand for binding pDNA and (ii) a support material allowing convection through the first chromatographic material, the liquid sample being derived from a cell culture harvest and having been subjected to a step of removing RNA prior to step (a); (b) eluting the liquid sample from the first chromatographic material comprising a purified mixture of supercoiled pDNA and open circular pDNA; (c) applying the liquid sample from step (b) to a second chromatographic material comprising a ligand that binds pDNA and allows selective separation of supercoiled pDNA from open circular pDNA; and (d) eluting the purified supercoiled pDNA from the second chromatographic material, the supercoiled pDNA eluted in step (d) having a purity of at least 95% without the use of any additional chromatographic material beyond the first and second chromatographic materials. Steps (a)-(d) and any intermediate steps can be completed within 5 hours. Uses of the supercoiled pDNA obtained by the separation method are further disclosed.

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 invention relates to the technical field of biology, in particular to a purification method and application of plasmid DNA. The plasmid DNA purification method comprises the following steps: mixing an inorganic salt and / or an organic solvent with plasmid DNA to obtain a sample; after balancing the chromatographic column by the equilibrium buffer solution, loading the sample obtained in the step S1 onto the chromatographic column; and performing primary elution on the chromatographic column by using the first buffer solution, and performing secondary elution on the chromatographic column by using the second buffer solution to obtain the purified superhelix plasmid DNA. According to the method, the plasmid DNA crude product is pretreated by adopting the inorganic salt and / or the organic solvent, so that the solubility of the plasmid DNA in the mobile phase in the purification process is reduced, the binding efficiency of a target object is further improved, and meanwhile, the loss of the plasmid DNA caused by flow-through of the plasmid DNA is avoided. By optimizing the purification process, the purpose of purifying the plasmid DNA only through one-step chromatography is achieved.

The invention discloses a nonlinear sliding mode superhelix control method for a multi-temperature-zone structure thermal test system, and relates to the field of aircraft complex environment simulation, and the method comprises the steps: building a time sequence heat transfer model of the multi-temperature-zone structure thermal test system based on the law of conservation of energy; defining a system state variable, and according to the system state variable, converting the time sequence heat transfer model of the multi-temperature-zone structure thermal test system into a state control equation; constructing a nonlinear sliding mode surface by using a state proportional term and an integral term of a thermal test system of the multi-temperature-zone structure; and introducing an adaptive superhelix reaching law to construct a nonlinear sliding mode superhelix controller of the thermal test system with the multi-temperature-zone structure. According to the nonlinear sliding mode superhelix control method for the multi-temperature-zone structure thermal test system, the problems of coupling, thermal radiation high-order terms, input variable heat loss fluctuation and the like of existing multi-temperature zones are solved, inhibition of concentrated disturbance is achieved, and the tracking precision of the multi-temperature zones is improved.

The invention discloses a method for increasing the yield of mRNA template plasmids produced by fermentation of a recombinant Stbl3 strain, which comprises the following steps: during fermentation culture of the recombinant Stbl3 strain, firstly inoculating a seed solution of the recombinant Stbl3 strain into a basic culture medium for fermentation culture, and then continuously adding the seed solution into a fed-batch culture medium for continuous fed-batch fermentation culture, the recombinant Stbl3 strain is prepared by transfecting Stbl3 competent cells with mRNA template plasmids, the basic culture medium and the fed-batch culture medium respectively contain betaine, and the carbon sources of the basic culture medium and the fed-batch culture medium are respectively glucose. According to the method, by optimizing a culture medium formula and fermentation condition control during fermentation culture and recombination of the Stbl3 strain, the yield of mRNA template plasmids produced by fermentation of the Stbl3 strain and the expression quantity of unit thalli are improved, and meanwhile, the monomeric superhelix proportion is also improved to a certain extent.

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.

A method, which synthesizes closed circular single-stranded and double-stranded DNA molecules using in vitro enzymatic systems, is described. Circular single-stranded DNA molecules and double-stranded DNA molecules (e.g., relaxed, or supercoiled) with various sizes can be synthesized. Unwanted DNA molecules, e.g., unligated oligomers, can be removed by exonucleases, such as T5 exonuclease, T7 exonuclease, lambda exonuclease, E. coli exonuclease I and / or III. A method of converting the single-stranded circular DNA molecules into double-stranded circular DNA molecules is also described. The single-stranded and double-stranded circular DNA molecules can be used in a variety of applications.

A conjugated composition and methods of using the same for treatment of a living cell, are provided. The conjugated composition includes a nucleic acid sequence directly or indirectly conjugated to at least one N-Acetylgalactosamine (GalNAc) molecule, or to a derivative of a GalNAc molecule. The conjugation is provided by a linker. The conjugated composition is structured to enable the at least one GalNAc molecule or derivative to deliver the nucleic acid sequence into a cell, for example, into a liver cell. The nucleic acid sequence can be a therapeutic supercoiled DNA minicircle that has a therapeutic gene for delivery to the cell.

A method, which synthesizes closed circular single-stranded and double-stranded DNA molecules using in vitro enzymatic systems, is described. Circular single-stranded DNA molecules and double-stranded DNA molecules (e.g., relaxed, or supercoiled) with various sizes can be synthesized. Unwanted DNA molecules, e.g., unligated oligomers, can be removed by exonucleases, such as T5 exonuclease, T7 exonuclease, lambda exonuclease, E. coli exonuclease I and / or III. A method of converting the single-stranded circular DNA molecules into double-stranded circular DNA molecules is also described. The single-stranded and double-stranded circular DNA molecules can be used in a variety of applications.

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.

Provided are new Cas proteins capable of gene editing with relaxed PAM requirement, or even does not require a PAM when targeting a negatively supercoiled double stranded DNA. It is further discovered that negatively supercoiled double stranded DNA in general reduces or even eliminates the PAM requirements for all Cas proteins. Accordingly, provided are compositions and methods for conducting gene editing, including base editing and prime editing, with relaxed or no PAM requirements.

An isolated monomeric supercoiled synthetic circular expression vector comprises a promoter operably linked to a gene of interest, preferably a gene useful in therapy wherein the synthetic vector comp

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.