3617 results about "Plasmid" patented technology

The embodiments of the present disclosure relate to decreasing the bioavailability of one or more target biomolecules by providing a composition that comprises a recombinant plasmid with one or more sequences of micro interfering ribonucleic acid (miRNA). When the recombinant plasmid interacts with a target cell, it causes the target cell to upregulate production of the miRNA, which then decreases the bioavailability of the target biomolecule. In some embodiments of the present disclosure, the target biomolecule is a cytokine or other mediator molecule of an inflammatory response.

The invention relates to the improvement of endonuclease-based antimicrobials by blocking DNA repair of double-strand break(s) (DSB(s)) in prokaryotic cells. In this respect, the invention especially concerns a method involving blocking DNA repair after a nucleic acid has been submitted to DSB, in particular by a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated programmable double-strand endonuclease. The invention particularly relates to the use of an exogenous molecule that inhibits DNA repair, preferably a protein that binds to the ends of the double-stranded break to block DSB repair. The invention also relates to vectors, particularly phagemids and plasmids, comprising nucleic acids encoding nucleases and Gam proteins, and a pharmaceutical composition and a product containing these vectors and their application.

The invention relates to a recombinant escherichia coli THRS and a method for synthesizing L-threonine by using the recombinant escherichia coli THRS. The invention belongs to the technical field of bioengineering, and provides a recombinant escherichia coli THRS which overexpresses encoding genes of ppc, aspC, aspA, thrAmutBC, asd, rhtC, rhtA, asdtm and thrAmut, knockout an encoding gene of fnr and knockout an encoding gene of iclR, the recombinant escherichia coli THRS takes glucose as a substrate for fermentation, and the yield and conversion rate of L-threonine are obviously improved; the L-threonine strain has the advantages that the L-threonine strain does not contain exogenous plasmids, all modification sites are integrated onto a genome, higher stability is achieved, antibiotics do not need to be added into a fermentation culture medium of the strain, and the produced L-threonine product is free of antibiotic residues and higher in safety.

The invention provides a multielement polyA polynucleotide for improving mRNA (messenger ribonucleic acid) stability and protein expression and application of the multielement polyA polynucleotide. The invention firstly provides a polynucleotide, the polynucleotide comprises more than two sections of polyA sequence fragments, the adjacent polyA sequence fragments are connected through Linkers, each polyA sequence fragment is composed of one or more continuous A, and each Linker is composed of C and / or G. The polynucleotide provided by the invention can improve mRNA stability and protein expression, and compared with a traditional unit poly-A sequence, the polynucleotide provided by the invention can greatly reduce the recombination rate of fermentation in escherichia coli so as to more easily optimize and amplify the process, and can be better applied to plasmid fermentation related to transcription template preparation in mRNA vaccine or drug production.

The invention belongs to the technical field of microorganisms, and particularly relates to recombinant escherichia coli containing a PDRN sequence and a preparation method thereof, and the preparation method comprises the following steps: S1, preparing a PDRN solution; s2, complementing the double strands of the PDRN into blunt ends; s3, constructing a recombinant plasmid; s4, carrying out mixed culture on a product obtained in the step S3 and escherichia coli DH5alpha; s5, screening the recombinants; s6, carrying out PCR (Polymerase Chain Reaction) amplification, and recording a strain from a fragment with the length of more than 200bp; s7, preparing a seed solution by adopting the strain prepared in the step S6; s8, strain fermentation; and S9, centrifugally collecting thalli. The method comprises the following steps: constructing a recombinant plasmid containing a PDRN fragment, introducing the recombinant plasmid into Escherichia coli, subsequently culturing the recombinant Escherichia coli to copy DNA in the Escherichia coli, extracting a purified exogenous gene fragment to obtain a strain containing a single fragment PDRN sequence, and getting rid of dependence on raw materials during subsequent extraction.

The invention belongs to the technical field of biology, and relates to an escherichia coli engineering strain for producing L-isoleucine through a methyl malic acid way as well as a construction method and application of the escherichia coli engineering strain. According to the invention, Escherichia coli BW25113 is used as a chassis strain, and a methyl malic acid synthase high-activity mutant gene cimA3.7, an isopropyl malic acid isomerase gene GsleuCD, a 3-isopropyl malic acid dehydrogenase gene AfleuB and a leucine dehydrogenase gene Lsleudh are over-expressed; a methyl malic acid absorption protein gene is over-expressed, and a branched chain amino acid transport system is modified to improve the exosome of the L-isoleucine; all methyl malic acid pathway related genes are integrated to a genome of a chassis strain, and a plasmid-free L-isoleucine production strain is constructed; the copy number of a key gene on a genome is optimized, and the yield of the engineering strain L-isoleucine is increased by introducing a non-oxidative glycolysis approach. The yield of the L-isoleucine after the engineering strain is fermented for 34 hours in a fermentation tank is 56.6 g / L.

The present invention relates to the construction of a high-yield indigo genetic engineering strain and a fermentation method. The present invention uses Escherichia coli as a starting strain, knocks out at least one of the yddG gene, trpR gene, tyrA gene or pheA gene in the host cell; integrates the tryptophanase gene tnaA and / or the flavin monooxygenase gene MaFMO on the genome; integrates one or more genes related to biofilm modification on the genome, and obtains the optimal engineering strain by comparing the fermentation yield; then transfers a plasmid into the strain to further strengthen the tryptophanase gene tnaA and the flavin monooxygenase gene MaFMO; finally, the yield is significantly increased by supplementing carbon sources and substrates and adding surfactants. The present invention provides an indigo-producing genetic engineering strain and a fermentation method with good stability and high synthesis efficiency, providing a reference for the subsequent development of more advantageous engineering strains.

The invention relates to a halophilic bacterium plasmid based on a sucrose lethal gene. The invention also provides a method for obtaining engineering halophilic bacteria without plasmids by using the halophilic bacteria plasmids.

The invention relates to the field of enzyme engineering, in particular to modified xylanase, a DNA (deoxyribonucleic acid) molecule, a recombinant plasmid and a transfected cell. The modified xylanase protected by the application is formed by replacing a deletion segment of a receptor enzyme with a substitution segment, the receptor enzyme is wild type xylanase, the sequence of the receptor enzyme is SEQ ID NO: 1, the deletion segment comprises a carbohydrate binding module of the receptor enzyme, the substitution segment is a carbohydrate binding module of a donor enzyme, the donor enzyme is a carbohydrate active enzyme, and the donor enzyme is a carbohydrate active enzyme. The sequence of the donor enzyme is one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 5. The carbohydrate binding module of the carbohydrate active enzyme with relatively high activity is replaced into the receptor enzyme, so that the binding capacity of the receptor enzyme and the carbohydrate is improved, and the activity of the receptor enzyme is improved.

The invention discloses construction and optimization of a gene editing tool based on CRISPR (clustered regularly interspaced short palindromic repeats)-associated transsposases and application of the gene editing tool in escherichia coli, and belongs to the field of synthetic biology and gene engineering. According to the invention, an efficient gene editing system is established in Escherichia coli, and the burden of a MUSCULAR-CAST system on strains is reduced through element optimization; by eliminating the self-targeting effect, large-fragment (> 10k bp) target gene expression cassettes can be efficiently integrated, and the editing degree of the gene editing system is improved. According to the invention, a MUSCULAR-CAST gene editing system is used for constructing the chassis strains for producing 3-fucosyllactose, L-tryptophan, 2 '-fucosyllactose and neotetraose, so that the chassis strains have equivalent or higher expression yield compared with plasmids, and the growth condition is better or the conversion rate is higher.

The invention relates to a chitosanase mutant capable of changing the polymerization degree of a product, a plasmid, a recombinant bacterium and application of the chitosanase mutant, and belongs to the technical field of enzyme engineering, the mutant is Csn-BJ-A134C or Csn-BJ-Q97K, the amino acid sequence of the mutant Csn-BJ-A134C is SEQ ID NO: 1, the amino acid sequence of the mutant Csn-BJ-Q97K is SEQ ID NO: 2, and the amino acid sequence of wild chitosanase Csn-BJ is shown as SEQ ID NO: 3. The invention also provides a recombinant vector, a recombinant bacterium and an enzyme preparation containing the mutant gene, and application of the recombinant vector, the recombinant bacterium and the enzyme preparation. The mutant disclosed by the invention is derived from GH46 family chitosanase Cs-BJ, molecular mutation modification is performed on the chitosanase, so that the catalytic activity of the chitosanase is improved, the polymerization degree of a final product chitosan oligosaccharide is changed, the polymerization degree of a minimum hydrolysis substrate is increased, and a foundation is laid for industrial application of the mutant.

The invention provides recombinant IV-type collagen as well as a preparation method and application thereof, and relates to the technical field of biochemical engineering. The preparation method of the recombinant IV-type collagen comprises the following steps: obtaining a codon from amino acid of the recombinant IV-type collagen, further obtaining a target gene, and synthesizing the target gene onto an expression vector to obtain a plasmid; transferring the plasmid into an expression host, and screening a positive transformant to obtain an expression strain; and culturing the expression strain, and expressing the target protein to obtain the crude recombinant IV-type collagen. The high-purity recombinant IV-type collagen is prepared through a genetic engineering technology, and the recombinant IV-type collagen can significantly down-regulate inflammatory factors and reduce inflammation; heat injury can be reduced by up-regulating heat shock protein; after heat injury, the recombinant IV-type collagen can obviously lower metal matrix protease of heat injury cells, and also can obviously promote gene expression of type I collagen and type III collagen, improve skin elasticity and moisture content and delay skin aging.

The invention discloses a P450 mutant and application thereof in synthesis of calcifediol, and belongs to the technical field of bioengineering. According to the present invention, the P450 enzyme mutant enzyme is constructed, and the single plasmid three-enzyme co-expression system of the redox partner and the cofactor cycle enzyme is constructed, such that the sufficient electron transport chain is provided for the P450 enzyme, and the calcifediol can be efficiently synthesized under the mild condition by using the vitamin D3 as the substrate; the conversion rate of calcifediol can reach 63.1%, and the conversion rate of the reaction is greatly improved. By adopting the method disclosed by the invention, the catalytic efficiency of the catalyst is improved, and the reaction cost is reduced.

Disclosed is an innovative method for genetic transformation through injection of Agrobacterium containing plasmid DNA expressing PLETHORAs (PLT5) or / and WOUNDINDUCED DEDIFFERENTIATION 1 (WIND1) in multiple plant species including Snapdragon and tomato. In addition, this plasmid has also been tested that it can significantly improve the genetic transformation method is not genotype dependent. Genetic transformation can be achieved by promoting de-novo regeneration of shoots in adult plants, which will of significant impact on some perennial plant species such as citrus and blue berry etc. The transgenic de-novo shoot regenerated from adult plants can by-pass the juvenile phase of plants regenerated through the tissue culture method. Expression of PLT5, WIND1, or both can also greatly improve plant regeneration and transgenic transformation through in-vitro tissue culture in recalcitrant plant species.

The invention provides a method for preparing a chicken immortalized cell line. The method comprises the following steps: constructing a plasmid system of conditional over-expression TERT (Telomerase Reverse Transcriptase), carrying out ovarian injection and breeding to obtain a transgenic chicken of the conditional over-expression TERT, and carrying out separation and induction to obtain the chicken immortalized cell line. The obtained chicken immortalized cell line has the characteristics of primary cells and is susceptible to viruses; the method can be used for virus reproduction and detection, influenza virus vaccine production and the like, can obtain immortalized cell lines of all organs and all parts of the chicken in a short time, and has a good application prospect in the vaccine industry.

The invention discloses a method for targeted creation of a fast-growing new tilapia strain, and belongs to the technical field of fish cultivation. The method specifically comprises the following steps: (1) taking a fragment, as shown in SEQ ID NO.1, on a third exon of a pomc gene as a gRNA target sequence; (2) designing and synthesizing a gRNA primer sequence; (3) amplifying by adopting the gRNA primer sequence and the gRNA plasmid, and then transcribing and purifying to obtain a gRNA fragment; (4) transferring the gRNA fragments into fertilized eggs of tilapia mossambica through gene editing to obtain F0-generation positive fish; and (5) hybridizing the F0-generation positive male fish with the wild-type female fish, screening out F1-generation parent fish, and culturing the F1-generation parent fish to obtain F2-generation tilapia with homozygous deletion mutation. According to the invention, the pomc homozygous knockout line is established in the Nile tilapia mossambica for the first time, the new fast-growing line is a complete gene function deletion model, the cost is lower, the efficiency is higher, and the phenotype can be stably inherited.

The preparation method comprises the following steps: firstly, analyzing the amino acid sequence of an alpha 1 chain of the human XVII type collagen, and selecting partial amino acid fragments which are not easily degraded by endogenous or exogenous protease in non-transmembrane regions of different functional domains of the protein; the recombinant collagen gene fragment (Col17a) n is obtained by repeatedly splicing the recombinant collagen gene fragment (Col17a) n; then introducing homologous promoters from different methylotrophic yeasts to the 5'end of a recombinant collagen gene segment, optimally synthesizing selected (Col17a) n and promoter segments, obtaining a connecting segment by utilizing PCR cloning, connecting the collagen segment (Col17a) n, the promoter segments and a pPIC9K carrier skeleton segment by virtue of a Gibson Assessment seamless connection technology, and obtaining the recombinant collagen gene. And constructing to obtain the pichia pastoris expression plasmid pPIC9K-(Col17a) n of the recombinant human source XVII type collagen.

The invention provides a 5-hydroxytryptamine Escherichia coli production strain and a construction method and application thereof, the strain is deleted with tnaA, pykA, trpR, ldhA and trpL genes, trpEfbr, aroGfbr, serAfbr, tktA and prsA genes are up-regulated, gdh and glnAfbr genes are heterologously expressed, a pykF gene is dynamically regulated and controlled, and meanwhile, the production strain carries a plasmid pACYC-FYSPD and a plasmid pETDuet-TM2-RBS-AnTDC; the constructed strain has the characteristic of metabolic balance, the expression levels of a tryptophan hydroxylation-decarboxylation system and a coenzyme tetrahydrobiopterin synthesis regeneration system are adjusted by optimizing the expression intensity of key genes of each precursor and simultaneously by virtue of a double-plasmid system, and 5-hydroxytryptamine can be efficiently synthesized in a culture medium without byproducts.

The invention relates to the technical field of microbial genetic engineering, in particular to a method for improving cordycepin synthesized by saccharomyces cerevisiae and saccharomyces cerevisiae engineering bacteria. According to the invention, codon-optimized 2 '-carbonyl-3'-deoxyadenosine reductase gene and 3 '-adenosine monophosphate phosphohydrolase gene from cordyceps militaris are expressed by using plasmids, and then the plasmids are transformed into host cells to obtain the saccharomyces cerevisiae engineering bacteria. And inoculating a seed solution obtained by seed culture of the activated saccharomyces cerevisiae engineering bacteria into a fermentation culture medium containing a metabolic effector, and carrying out fermentation culture to synthesize cordycepin. Metabolic effectors (Cu < 2 + >, Fe < 2 + >, Mg < 2 + >, Zn < 2 + >, citric acid, cysteine, aspartic acid, glycine, VB1 and tea polyphenol) are added into a fermentation culture medium strain, and the variety and concentration of the metabolic effectors are optimized, so that the yield and synthesis efficiency of cordycepin are remarkably improved. By using the combination of the strain obtained by the invention and the optimal effector, the yield of cordycepin in a fermentation tank reaches 2.9 g / L.

The invention discloses a method for aided analysis of influence of 5 'UTR on mRNA translation efficiency based on a machine learning model, and relates to the technical field of synthetic biology and computational biology, the method comprises the following steps: S1, constructing a 5' UTR plasmid library; s2, transcribing the 5 'UTR plasmid into an independent mRNA (messenger ribonucleic acid) with a luciferase-opt luciferase, and taking the luminous intensity of the luciferase-opt luciferase as the translation intensity of the mRNA; s3, analyzing characteristics related to protein expression intensity of the mRNA; and S4, constructing a random forest algorithm model on the basis of the features analyzed in the step S3 to predict the influence of the 5 'UTR on the mRNA translation efficiency. According to the method for auxiliary analysis of the influence of the 5 'UTR on the mRNA translation efficiency based on the machine learning model, a feasible technical route reference is provided for sequence optimization of mRNA in subsequent mRNA vaccine development, so that the mRNA vaccine can efficiently express target protein in a body.

The invention relates to a method for induced differentiation of skeletal muscle cells by porcine embryonic stem cells. Comprising the following steps: (1) constructing a PAX7-overexpressed porcine embryonic stem cell stable cell line; (2) induced differentiation of skeletal muscle cells; (3) performing immunofluorescence identification; wherein the step (1) specifically comprises the following steps: 1.1, constructing a DOX (deoxyribonucleic acid) induced PAX7 expression plasmid PiggyBac-TRE3G-EF1a-PAX7; 1.2, performing electrical transformation on the porcine embryonic stem cells cultured for two days, and adding Buffer E into an electrical transformation cup; taking Buffer R, mixing plasmid and PBase, uniformly mixing, and re-suspending cell precipitate; 1.3, after electrotransformation is finished, the cells are planted on the feeding layer, Y-27632 is added into the culture medium, and the culture medium without Y-27632 is replaced with the culture medium without Y-27632 on the second day; and 1.4, after culturing for 3-4 days, selecting the porcine embryonic stem cells which are successfully subjected to electrotransfection for amplification culture, and naming PAX7-PESCs.

The invention provides a method for constitutive expression of foreign protein by escherichia coli Nissle 1917, and belongs to the technical field of genetically engineered bacteria. The invention provides a method for constitutive expression of foreign protein by Escherichia coli Nissle 1917 (EcN), which is characterized in that EcN is used as a chassis strain, an engineering strain for constitutive expression of the foreign protein is constructed by transforming a covert plasmid, and high-efficiency expression of the foreign protein can be realized without induction of an inducer and antibiotic pressure. By utilizing the method disclosed by the invention, the EcN engineering bacterium for constitutive expression of the foreign protein RtSOD is successfully constructed, constitutive efficient expression of the high-stability RtSOD protein in the EcN engineering bacterium can be realized without using antibiotic pressure and inducer induction, and the EcN engineering bacterium can be used as a potential viable bacterium medicine for preventing and treating oxidative stress related diseases and has a wide application prospect. And the method has important significance on the practical application of the EcN engineering strain.

The invention provides a lentivirus for treating cancer as well as a preparation method, application and a corresponding pharmaceutical composition of the lentivirus. According to the invention, an hTERT promoter and a CDC6shRNA gene sequence are inserted behind an enzyme cutting site of a vector, an obtained plasmid is transferred into an expression strain, the expression strain is cultured and screened to obtain a cloned strain, and the cloned strain is shaken and packaged to obtain the hTERT-CDC6shRNA lentivirus; when the lentivirus disclosed by the invention is used for infecting cancer cells, the experimental result shows that the proliferation of the cancer cells can be effectively inhibited.

The invention relates to a gene editing vector of agrobacterium, a gene editing method of the gene editing vector and an agrobacterium editing strain. The agrobacterium gene editing vector comprises the following gene elements: a plasmid skeleton, a Pvan promoter, a spCas9 gene, a green fluorescent protein gene GFP, gRNA, and an upstream homologous arm and a downstream homologous arm of a target gene. According to the agrobacterium gene editing vector, a CRISPR / spCas9-GFP gene editing system is adopted, an efficient, simple, convenient and traceless genome editing system is constructed in agrobacterium, and gene knockout and insertion can be achieved.

The present invention relates to a method for finding global regulators in order to induce or increase production of target secondary metabolites. The method comprises providing a bacterium indicator strain producing two fluorescent reporter signals under the same control of two different biosynthetic gene clusters (BGC), preferably highly expressed BGC, performing random mutagenesis in said bacterium indicator strain, and selecting mutant bacteria not producing said reporter signals. The invention also relates to a method to activate the production of target secondary metabolites, and to a CRISPR / Cas based single plasmid to delete or inactivate a global regulator and / or activate a BGC. Finally, the inventive method can be performed in high-throughput format allowing to speed-up the procedure.

A stable transformant, in which a gene of interest is incorporated into the genome, is simply and efficiently produced.A method for producing a transformant, comprising a step of introducing into a host, linear genome-introduced nucleic acid fragment(s) comprising a gene of interest and a helper plasmid for transformation having a pair of homologous recombination sequences for incorporation of the linear genome-introduced nucleic acid fragment(s), and then selecting a transformant, in which the gene of interest is incorporated into the predetermined position in the host genome and the gene of interest is expressed therein.

The invention relates to the technical field of protein interaction in yeast, in particular to a yeast double-expression vector, a construction method thereof, a protein interaction identification method and application. According to the vector, two exogenous genes can be cloned in the same vector, and the two exogenous genes can be expressed in yeast at the same time, so that interaction identification can be carried out. The work of plasmid construction and extraction, library preparation, yeast hybridization, screening culture and the like of an original yeast two-hybrid system is greatly reduced, and the identification efficiency of protein interaction is remarkably improved.

The invention relates to the technical field of engineering bacteria, in particular to conjugational transfer receptor bacteria and a construction method and application thereof. The invention provides a conjugational transfer receptor bacterium, and the conjugational transfer receptor bacterium is Escherichia fergusonii M112, is preserved in the China Center for Type Culture Collection (CCTCC), and has the preservation number of CCTCC NO: M20242751. The conjugational transfer acceptor bacterium provided by the invention is stable in character, is simple to operate as a conjugational transfer acceptor, has sodium azide resistance, and can be used as an acceptor bacterium to detect the conjugational transfer efficiency of donor bacterium plasmids under the screening condition of sodium azide.

The present application relates to a virus of reverse genetic operation construction of NADC34-like PRRSV2. Specifically, the full gene sequence of NADC34-like PRRSV2 BJ1805-2 isolated strain is segmented and connected to the vector by using PCR amplification, enzyme digestion and other methods with pACY177 plasmid as the carrier, and the BJ1805-2 full-length infectious clone recombinant plasmid (rBJ1805-2) is obtained. The infectious clone virus rBJ1805-2 is rescued in vitro, and the first NADC34-like PRRSV2 strain infectious clone platform in China is successfully built. The present application also relates to a modification method for constructing NADC34-like PRRSV2 strain which can adapt to Marc-145 cell passage culture. The infectious clone virus constructed by the present application has good in vitro and in vivo proliferation efficiency, and can cause specific viremia after inoculation in pigs; it has good safety, does not cause fever after inoculation in pigs, and will not cause piglet death. The modified strain can be used as a candidate strain for developing the first NADC34-like PRRSV2 specific vaccine in China, and is conducive to the prevention and control of PRRSV epidemic in China.

The invention discloses construction and application of an escherichia coli L-tryptophan biosensor, and belongs to the technical field of bioengineering. The L-tryptophan biosensor provided by the invention comprises a mutated leader peptide fragment tnaC-tnaA, a promoter of the mutated leader peptide fragment tnaC-tnaA, a green fluorescent protein coding gene eGFP and a pTrc99a plasmid skeleton, a recombinant strain containing the L-tryptophan biosensor is cultured, the expression of green fluorescent protein in a culture solution is gradually enhanced along with the increase of the concentration of the L-tryptophan, and the fluorescence value of a unit cell shows better correlation when the concentration of the L-tryptophan is 0-1.5 g / L; therefore, when the biosensor is used for detecting the L-tryptophan in the fermentation liquor, the biosensor has the characteristics of simplicity in operation, sensitive response and high threshold value.