23 results about "Plasmid recombination" patented technology

The invention relates to the technical field of genetic engineering, in particular to a recombinant vector, an expression plasmid, a recombinant schizochytrium limacinum mutant and a method for increasing the DHA content of microorganisms. The LIP1 gene for coding lipase in the microbial lipid droplet protein is knocked out through a homologous recombination technology, and the production of lipase in the microbial lipid droplet can be effectively inhibited, so that the hydrolysis process of triglyceride of polyunsaturated fatty acid stored in the lipid droplet to glycerol and free fatty acid in the later stage of microbial culture is blocked, and DHA is prevented from being lost from a TAG repository; experimental verification shows that in microorganisms represented by schizochytrium limacinum, the DHA content can be increased by 21.8% compared with that of a wild type, the yield and purity of DHA are increased, the stability of the microbial fermentation process is guaranteed, and application and popularization are facilitated.

The invention relates to a phospholipase D mutant Lip-Gri / NQ, a plasmid, a recombinant bacterium and application of the phospholipase D mutant Lip-Gri / NQ and belongs to the field of enzyme engineering, the amino acid sequence of the phospholipase D mutant is shown as SEQ ID NO: 1, and the nucleotide sequence of a gene for coding the phospholipase D mutant Lip-Gri / NQ is shown as SEQ ID NO: 2. The invention also provides a recombinant plasmid and an engineering bacterium containing the gene of the recombinant plasmid phospholipase D mutant Lip-Gri / NQ, and application of the phospholipase D mutant Lip-Gri / NQ in catalyzing phospholipids to generate specific-configuration functional lipids including phosphatidylserine. According to the phospholipase D Lip-Gri disclosed by the invention, wild phospholipase D Lip-Gri from Streptomyces griseofuscus is transformed through site-directed mutagenesis, and the enzyme activity of the obtained mutant is remarkably improved and is 11 times that of the wild phospholipase D Lip-Gri.

The invention discloses a recombinant plasmid, a recombinant strain, recombinant phycocyanin and application of the recombinant plasmid, the recombinant strain and the recombinant phycocyanin in mercury ion detection. The invention further discloses a dual-mode mercury ion biosensor based on the thermal-stability recombinant phycocyanin and a preparation method and application of the dual-mode mercury ion biosensor. The biosensor based on the recombinant phycocyanin has specific fluorescence response to trace mercury ions. The recombinant phycocyanin pigment binding rate is higher than that of all reported recombinant phycocyanin, great Stokes shift (288 nm) is shown, the mercury ion selectivity is high, the thermal stability is good, the prepared biosensor shows the characteristics of dual-mode output and ultra-sensitive sensing, the detection limit under a fluorescence-based mode is 2.94 nM, and the biosensor has good application prospects. And the detection limit is 1.87 nM in a colorimetric mode based on fluorescence. The protein-based sensor is obtained from escherichia coli, is simple and convenient, supports in-situ monitoring of a smart phone, and is low in cost, excellent in performance and free of any toxicity.

The invention relates to the technical field of biology, in particular to a Zymomonas mobilis terminator library as well as a construction method and application thereof. Through terminator prediction, primer design, sequencing analysis and efficiency calculation, the stable and efficient Zymomonas mobilis terminator library with high robustness is obtained through screening. The terminator sequence in the library is low in repeatability and high in matching degree with a known transcription termination site. The library constructed by the invention can effectively avoid plasmid recombination, can be directly used for optimizing expression of key enzymes in a metabolic pathway, improves metabolic flux by enhancing mRNA stability, and provides an efficient tool for metabolic engineering modification of Zymomonas mobilis.

The invention provides a method for improving the recombination efficiency of denitrifying hyphomicrobe and application, and belongs to the technical field of gene engineering. On the basis of a pK18mobsacB-Tc vector, a vector capable of simultaneously expressing exonuclease-recombinase and upstream and downstream homologous arms of a target gene is constructed, the vector is transformed into denitrifying hyphomicrobe for homologous recombination, mediation of other plasmids is not needed, traceless knockout of the target gene can be completed only through one round of transformation, and the method is simple and convenient to operate. Compared with existing gene knockout plasmids and transformation methods of denitrifying hyphomicrobe, the method is more efficient. Experiments prove that when the vector is used for constructing a denitrifying hyphomicrobium engineering strain, compared with plasmids without recombinase, the number of recombinants is remarkably increased, and the vector has the characteristics of simplicity, convenience and high efficiency.

The present application relates to a kind of chitosanase mutant WP-E80A, plasmid, recombinant bacteria and its application, belong to enzyme engineering technical field, the amino acid sequence of the chitosanase mutant as shown in SEQ ID NO:1, the amino acid sequence of wild-type chitosanase CsnWP as shown in SEQ ID NO:2.Compared with wild-type chitosanase CsnWP, the 80th amino acid is subjected to site-directed mutagenesis.The present application also provides plasmid and recombinant engineering bacteria comprising the chitosanase mutant WP-E80A gene, and the chitosanase mutant WP-E80A can be prepared to generate chitotriose with chitobiose as substrate.

The application discloses a recombinant adenovirus shuttle plasmid and a preparation method thereof, a recombinant adenovirus and application thereof, and a Zika virus recombinant adenovirus plasmid, and belongs to the field of biological medicines. The Zika virus recombinant adenovirus shuttle plasmid provided by the application takes pShuttle as a carrier, carries a Zika virus E protein mutant gene, and the 5' end of the Zika virus E protein mutant gene is connected with a signal peptide gene; the 5' end of the signal peptide gene is connected with a Kozak sequence; and the 5' end of the Kozak sequence is connected with an enzyme cutting site 1. The recombinant shuttle plasmid, the recombinant plasmid and the recombinant adenovirus provided by the application can be applied to the preparation of a Zika virus adenovirus vaccine, and have wide development and application prospects.

Provided herein are recombinant self-complementary adeno-associated virus (scAAV) nucleic acid vectors containing ubiquitous eukaryotic promoters, such as elongation factor 1 alpha (EF1α), chicken beta-actin (CBA), and hybrid chicken beta-actin (CBh), followed by a dominant-negative RhoA. Also provided herein are methods of using the vectors, including intraocular injection (e.g., intracameral injection) to reduce intraocular pressure (IOP). Also provided herein are plasmids, recombinant scAAV particles, compositions, formulations, and other methods of use related to such vectors.

The invention discloses plasmid-free recombinant escherichia coli for synthesizing lactoyl-N-neotetraose as well as a construction method and application of the plasmid-free recombinant escherichia coli, and belongs to the field of gene engineering. An engineering strain MG-LNT-07 is used as a starting strain, beta-1, 4-galactosyl transferase Hplex2B is introduced, the copy number of the engineering strain is optimized, a multidrug efflux transporter gene mdfA is integrated, the expression of a glycoside donor pathway gene is fine-tuned, and a rapZ gene is knocked out, so that a plasmid-free strain for efficiently synthesizing the lactoyl-N-neotetraose is constructed, finally, the extracellular titer of LNnT is 7.76 g / L, and the expression of the lactoyl-N-neotetraose can be efficiently synthesized. And only 0.58 g / L of precursor LNTriII is remained, so that the method has a good industrial prospect.

The invention belongs to the technical field of recombinant proteins, and particularly relates to a gene for coding a split-ring loganin synthase, the split-ring loganin synthase, a recombinant plasmid, a recombinant bacterium and an application of the split-ring loganin synthase in catalyzing loganin acid to generate a split-ring iridoid compound, wherein the gene is cloned and separated from a gene in gentiana macrophylla pall. The sequence of the gene for coding the split-ring loganin synthase is as shown in SEQ ID NO.01, and the amino acid sequence of the split-ring loganin synthase is as shown in SEQ ID NO.02. The recombinant plasmid contains a sequence shown in SEQ ID NO. 01. The recombinant bacteria comprise recombinant yeast WAT11 or recombinant agrobacterium GV3101 and the like containing recombinant plasmids. The ring-splitting loganin synthase produced and expressed by the recombinant yeast WAT11 can be used for catalyzing loganin acid to generate sweroside, gentiopicroside or ring-splitting loganin acid. The split-ring loganin synthase produced and expressed by the recombinant agrobacterium GV3101 catalyzes loganin acid in tobacco leaves to produce gentiopicroside.

The present application relates to a kind of chitosanase mutant CsnW27-HF, plasmid, recombination bacteria and its application, belong to enzyme engineering technical field, the amino acid sequence of the chitosanase mutant as shown in SEQ ID NO:1, the amino acid sequence of wild-type chitosanase CsnW27 as shown in SEQ ID NO:2.Compared with wild-type chitosanase CsnW27, the 82th and 99th amino acid are subjected to site-directed mutagenesis.The present application also provides plasmid and recombination engineering bacteria comprising the chitosanase mutant CsnW27-HF gene, the enzyme activity of the mutant is increased by 2.36 times compared with wild type.

This application relates to the field of biotransformation technology, specifically to a transaminase mutant, recombinant plasmid, recombinant bacterial strain, its application, and synthesis method. The transaminase mutant is obtained by mutating a wild-type transaminase, which originates from heterotrophic Gram-negative bacteria and includes the amino acid sequence shown in SEQ ID NO. 5. The mutation sites of the transaminase mutant include any one or more of M439I, V419A, A239P, T276S, H310E, T36A, R43K, N48T, and A165S. By performing site-directed mutagenesis on the wild-type transaminase, a transaminase mutant with excellent overall enzyme activity is obtained and applied to the biocatalytic synthesis of 1,6-hexanediamine, significantly increasing the yield of 1,6-hexanediamine, reducing its industrial production cost, and improving production efficiency.

The present invention provides isolated plasmids, recombinant microorganisms, kits, and methods for the treatment of inflammatory skin disease.

The invention belongs to the technical field of gene engineering, and relates to a recombinant plasmid, a recombinant system and a construction method and application thereof. The plasmid expresses an operon under the induction of a promoter, and the operon is composed of a CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-associated 9) system and a Red / ET recombinase system derived from pseudoalteromonas P. agarivorans Hao 2018. The recombinase system comprises a recombinase 2018Red [beta] and a recombinase 2018Red [alpha]; homologous arms with different lengths for recombination are inserted between the Cas9 protein and the recombinase 2018Red alpha beta. According to the invention, the homologous arm and the guide RNA sequence are constructed to the same site of the plasmid, and the construction can be completed by only one step of Gibson assembly, so that the workload is reduced, the working efficiency is improved, and the gene editing period can be completed in a relatively short time. The invention provides a new tool for gene editing, and has a wide application prospect.

This invention discloses a dual-plasmid recombinant Escherichia coli engineered bacterium, its construction method, and its applications, belonging to the fields of synthetic biology and immunotherapy. Using the safe probiotic Escherichia coli Nissle 1917 as the chassis strain, this invention constructs a recombinant Escherichia coli engineered bacterium through heterologous expression of the TNF-IL23R fusion protein via a dual-plasmid system and the inclusion of a quorum sensing-regulated lysis circuit. This recombinant engineered bacterium can efficiently express the TNF-IL23R fusion protein, simultaneously blocking the TNF-α and IL-23 dual pathways, enhancing anti-inflammatory effects and reducing the risk of drug resistance, thus achieving therapeutic effects. It can also secrete N-acylhomoserine lactone, utilizing the quorum sensing system to regulate lysis genes, controlling bacterial population density, avoiding excessive bacterial population burden on the body, and simultaneously achieving steady drug release by controlling bacterial autolysis. This provides a high-quality bacterial strain resource for in vivo therapy of autoimmune diseases.

This invention discloses a linearized mutant plasmid of Tembusu virus SLII, a recombinant virus, and its applications, belonging to the field of molecular biology. Using the pACNR CQW1-Intron infectious clone as a backbone, the SLII stem-loop structure within the 3′UTR Domain-I region was linearized to construct the SLII linearized mutant infectious clone pACNR-CQW1-SLIILiner, which was then rescued to obtain the recombinant virus CQW1-SLIILiner. This recombinant virus exhibits limited replication capacity at the in vitro cellular level and displays a significantly attenuated phenotype in animal models. It can also induce specific neutralizing antibodies and cellular immune responses, providing significant protection against virulent strains. Therefore, the recombinant virus of this invention possesses advantages such as clear molecular genetic markers, high safety, good immunogenicity, and strong genetic stability, and can be applied to the preparation of live attenuated vaccines or related biological products for the prevention of TMUV infection.

The invention relates to the technical field of biotransformation, in particular to a transaminase mutant, a recombinant plasmid, a recombinant strain, application and a synthesis method. The transaminase mutant is obtained by mutating wild type transaminase, the wild type transaminase is derived from heterotrophic gram negative bacteria and comprises an amino acid sequence as shown in SEQ ID NO.5, and mutation sites of the transaminase mutant comprise any one or more of M439I, V419A, A239P, T276S, H310E, T36A, R43K, N48T and A165S. The transaminase mutant with excellent overall enzyme activity is obtained by performing site-directed mutagenesis on wild transaminase, and is applied to biocatalytic synthesis of 1, 6-hexamethylenediamine, so that the yield of 1, 6-hexamethylenediamine is remarkably increased, the industrial production cost of 1, 6-hexamethylenediamine is reduced, and the production benefit is improved.

The application relates to a phospholipase D mutant Lip-Gri / NQ, a plasmid, a recombinant bacterium and application thereof, belongs to the field of enzyme engineering, the amino acid sequence of the phospholipase D mutant is shown as SEQ ID NO: 1, the gene for encoding the phospholipase D mutant Lip-Gri / NQ is shown as SEQ ID NO. 2. The application also provides a recombinant plasmid containing the recombinant plasmid phospholipase D mutant Lip-Gri / NQ gene and an engineering bacterium, and application of the phospholipase D mutant Lip-Gri / NQ in catalyzing phospholipid to generate specific configuration functional lipids including phosphatidylserine. The application is obtained by site-directed mutagenesis on wild-type phospholipase D Lip-Gri, the enzyme activity of the mutant is significantly improved, and is 11 times that of the wild type. Streptomyces griseofuscus The enzyme activity of the mutant is significantly improved, and is 11 times that of the wild type.

The present disclosure provides isolated plasmids, recombinant microorganisms, kits, and methods for treating microbial infections, particularly MRSA. In a first aspect, the disclosure provides a recombinant microorganism comprising deletions or substitutions in one or more genes encoding D-alanine biosynthesis genes, where the D-alanine biosynthesis genes are inactive, deletions or substitutions in one or more genes encoding naturally occurring antibiotic resistance genes, where the naturally occurring antibiotic resistance genes are inactive, and deletions or substitutions in one or more genes encoding naturally occurring lysogenic bacteriophage genes, where the naturally occurring lysogenic bacteriophage genes are inactive.

The invention relates to a chitosanase mutant CsnW27-HF, a plasmid, a recombinant bacterium and application of the chitosanase mutant CsnW27-HF, and belongs to the technical field of enzyme engineering, the amino acid sequence of the chitosanase mutant is shown as SEQ ID NO: 1, and the amino acid sequence of wild chitosanase CsnW27 is shown as SEQ ID NO: 2. Compared with wild chitosanase CsnW27, the chitosanase CsnW27 has the advantage that site-specific mutagenesis is carried out on 82nd and 99th amino acids. The invention further provides plasmids and recombinant engineering bacteria containing the chitosanase mutant CsnW27-HF gene, and the enzyme activity of the mutant is improved by 2.36 times compared with that of a wild type.

The application provides a kind of composite protein, recombinant plasmid, recombinant escherichia coli and application.The composite protein includes nanometer compartment protein and the cargo protein loaded in nanometer compartment protein, nanometer compartment protein loads cargo protein through targeting peptide, the amino acid sequence of targeting peptide is as shown in Seq.ID No.1.The application first clearly defines DyP-linker as the key signal sequence of CFP-29 encapsulating cargo protein, by accurately retaining the core sequence, avoid the problem of blind modification caused by unclear signal sequence in the prior art.Target protein realizes targeted encapsulation through the specific interaction of DyP-linker and CFP-29 spherical shell protein, effectively solves the defects of low encapsulation efficiency and non-specific binding in traditional modification, ensures that the target protein can efficiently and stably enter the nanosphere shell.

The invention discloses sgRNA, an editing plasmid, a recombinant plasmid, an AviTag engineered bacteriophage, a magnetic bacteriophage probe, and a construction method and application thereof. The invention further discloses an enrichment separation and quantitative detection method for the pseudomonas aeruginosa. The magnetic bacteriophage probe can rapidly and specifically enrich pseudomonas aeruginosa in complex matrixes such as serum, infectious microbes and interference components are removed through magnetic separation, and sensitive quantitative detection of target bacteria is achieved in combination with an ATP bioluminescence system. According to the invention, by optimizing the AviTag-linker configuration, the display and biotinylation efficiency of the AviTag tag on the surface of the bacteriophage is remarkably improved, on the premise of keeping the titer of the bacteriophage and the stability of a host spectrum, the capture capability of the magnetic bacteriophage probe is enhanced, and low detection limit, short detection time and good repeatability are realized.