46 results about "Coli strain" patented technology

The invention discloses a method for synthesizing alpha-arbutin through biotransformation, and belongs to the technical field of biochemistry. A gene cloning technology is utilized, sucrose phosphorylase Spase in leuconostoc mesenteroides and sucrose phosphorylase gtfa in bifidobacterium adolescentis are screened and found through a Deep Molecules and DLKcat algorithm on a Uniprot database, novopro is utilized for codon optimization, then the sucrose phosphorylase Spase and the sucrose phosphorylase gtfa are expressed on a plasmid vector PET-28a and are converted into an escherichia coli BL21 (DE3) strain, and the sucrose phosphorylase Spase and the sucrose phosphorylase gtfa in the bifidobacterium adolescentis are obtained. An engineering strain for heterologous expression of the gene is constructed by taking the gene as a chassis cell. According to the process, sucrose and hydroquinone are taken as co-substrates, and bioconversion is carried out under the action of sucrose phosphorylase from different sources to obtain alpha-arbutin.

The invention provides a bacterial outer membrane vesicle drug carrier with a targeting effect and preparation, and relates to the technical field of biological medicines.The bacterial outer membrane vesicle protein ClyA derived from an escherichia coli W3110 strain is combined with iRGD protein capable of being combined with an integrin receptor alpha v beta 3, a ClyA-iRGD-pGEX-6P-1 recombinant plasmid for expressing ClyA-iRGD fusion protein is constructed, and the ClyA-iRGD-pGEX-6P-1 recombinant plasmid is used for preparing the bacterial outer membrane vesicle drug carrier with the targeting effect. By endogenous expression of the plasmid in W3110 escherichia coli, the C-iRGD-OMVs which has a tumor targeting effect and shows ClyA-iRGD fusion protein on the surface is prepared. The C-iRGD-OMVs shows efficient targeting binding capacity to MCF-7 breast cancer cells, overcomes the defect that iRGD single peptide is prone to aggregation and crystallization, has the potential of loading drugs, lays a foundation for the C-iRGD-OMVs serving as a targeting carrier for delivering breast cancer treatment drugs, and is expected to be further developed into a targeting treatment preparation for breast cancer.

The invention belongs to the technical field of biological gene engineering, and particularly relates to escherichia coli with high RNA content and a construction method and application thereof.A strong promoter is used for replacing a promoter of escherichia coli rDNA, an expression element of strong promoter + rDNA + terminator is constructed, plasmid is used as a carrier, the expression element is transferred into escherichia coli, and an escherichia coli engineering strain with high RNA yield is constructed; wherein the rDNA is derived from one or more of rrnA, rrnB, rrnC, rrnD, rrnE, rrnG and rrnH of rDNA operons of escherichia coli, and comprises 16S rDNA, 23S rDNA and 5S rDNA in the corresponding operons. By adopting the construction method, the RNA content of the escherichia coli can be obviously improved, and when the method is used for producing RNA, the RNA yield is obviously higher than that of an initial escherichia coli strain and common saccharomycetes for RNA production at present.

This disclosure provides an E. coli strain, which lacks thioredoxin reductase activity encoded by trxB and thioredoxin 1 activity encoded by trxA, and glutathione reductase activity encoded by gor. Said E. coli strain expresses a mutated AhpC protein having glutathione reductase activity and a cytosolic prokaryotic disulfide isomerase. The E. coli strain has an oxidative cytosol and can be used to efficiently produce proteins having disulfide bonds.

The invention discloses an escherichia coli system for synthesizing D-lactic acid as well as a preparation method and application of the escherichia coli system. The escherichia coli system comprises a strain GL10 and a strain XL15; the bacterial strain GL10 is obtained by deleting genes of ppsA, mgsA, dld, pta, poxB, pflB, adhE, frdA, frdB and frdC in an escherichia coli BL21 bacterial strain; the bacterial strain XL15 is obtained by deleting genes of glk, ptsI, ptsG, mglB and galP in a bacterial strain GL10. The strain GL10 efficiently utilizes glucose, and the strain XL15 utilizes xylose, so that the CCR effect is effectively relieved, efficient synergistic utilization of a mixed carbon source is realized, the yield and purity of D-lactic acid are improved, and the production cost is reduced.

The invention discloses a synthesis method of ubiquitin (1-74) NHNH2, and belongs to the technical field of protein synthesis. The synthesis method comprises the following steps: utilizing a prokaryotic expression system, expressing a full-length protein of Ub (1-76) and a Josephin protein coded by Burkholderia pyrrocinia by virtue of an escherichia coli BL21 (DE3) strain, hydrolyzing the Ub (1-76) by virtue of the obtained 6His-BpJOS, cutting a peptide bond between Ub Arg-74 and Gly-75, and capturing a generated thiolipid intermediate by virtue of hydrazine hydrate, so as to obtain the Ub (1-74) NHNH2. According to the present invention, the ubiquitin cleavage enzyme BpJOS catalyzed in-situ activation process is adopted, such that the preparation yield is high, the operation is simple, the large-scale preparation can be achieved, the synthesis cost is low, and the method can be used for synthesizing the multi-ubiquitination modified fragment and synthesizing the Ub (1-74) probe as the precursor, and has wide application prospects.

The invention discloses an escherichia coli strain with high yield of riboflavin as well as a construction method and application of the escherichia coli strain. A series of Escherichia coli strains with high yield of riboflavin are constructed, the constructed Escherichia coli with high yield of riboflavin is safe and harmless, glucose is used as a substrate for fermentation under the aerobic condition of a shake flask, the yield of riboflavin is 1117.8 mg / L, 1330.1 mg / L, 1430.8 mg / L, 1508.7 mg / L and 1640.8 mg / L in sequence, and the yields are 112.5 mg / g of glucose, 134.1 mg / g of glucose, 146.3 mg / g of glucose, 158.8 mg / g of glucose and 168.1 mg / g of glucose in sequence; when the strain SN04M is subjected to fed-batch fermentation in a 1L fermentation tank, the yield of riboflavin reaches 8.52 g / L, and the total yield is 189.9 mg / g glucose. According to the invention, the fermentation yield of riboflavin and the substrate utilization rate of riboflavin to glucose can be obviously improved.

The invention discloses an Escherichia coli strain capable of stably expressing near-infrared fluorescent protein as well as a construction method and application of the Escherichia coli strain, and belongs to the technical field of gene engineering. According to the bacterial strain, escherichia coli probiotics Nissle 1917 serve as a chassis bacterial strain, at least two miRFP713 gene expression cassettes are integrated on a chromosome through a CRISPR-Cas mediated homologous recombination technology, and each expression cassette comprises a constitutive promoter and a miRFP713 gene optimized by a codon; meanwhile, the biliverdin synthetase gene is integrated to realize cofactor self-supply. The chromosome integrative Escherichia coli strain constructed by the invention realizes stable expression without exogenous induction, the fluorescence signal retention rate of 20 generations of passage is greater than 98%, and the fluorescence intensity is improved by 94.8 times, and is suitable for multiple application scenes such as long-term living body tracking, tumor targeted tracking and the like.

The invention discloses an Escherichia coli strain capable of bidirectionally regulating the copy number of CoIE1 type plasmids and application of the Escherichia coli strain. According to the method, escherichia coli Mach1-T1 is taken as an initial strain, a CRISPR / CAS9 gene editing technology is utilized, RNase E Q36R variant genes, rssB genes and pcnb genes are constructed into a polycistron expression cassette, and the polycistron expression cassette is placed under the control of an arabinose operon to replace endogenous pcnb genes of a genome. Each gene in the polycistron expression cassette is driven to translate by an independent ribosome binding site (RBS). By adjusting the concentration of the arabinose, continuous and bidirectional regulation and control of the copy number of the CoIE1 type plasmids from'inhibition 'to'overproduction' can be realized, and the method has important significance on industrial mass production of the CoIE1 type plasmids and diversified application of the CoIE1 type plasmids.

The invention discloses Escherichia coli for fermentation production of riboflavin anti-bacteriophage, and belongs to the technical field of microorganisms. Receptor genes of bacteriophages are excavated through co-evolution of bacteriophages T1, T7, vBEcoMP251 and vBEcoMP255 and escherichia coli strains, the resistance of escherichia coli to the bacteriophages is enhanced by knocking out screened bacteriophage receptors on the genome level, and meanwhile, defense systems Gabija and KELM with the ability of resisting bacteriophages JNUWH1 and JNUWD and defense systems Shedu and Septu with the ability of resisting bacteriophages T4 are obtained through screening. Gene is integrated to a genome to strengthen the resistance of escherichia coli to bacteriophage, construction of the anti-bacteriophage chassis cell is completed, meanwhile, genes lacI, pfkA, edd and eda are knocked out, the anti-bacteriophage escherichia coli chassis cell for fermentation production of riboflavin is obtained, and a certain application prospect is achieved.

The invention belongs to the technical field of genetic engineering, and discloses a genetically engineered bacterium for efficiently synthesizing gamma-aminobutyric acid from the beginning by efficiently utilizing a mixed carbon source, a method and application, and the genetically engineered bacterium is obtained by overexpressing edd from Zymomonas mobilis on the basis of escherichia coli E. coli G16; eda from the zymomonas mobilis is over-expressed; performing overexpression on acs (Acetobacter pasteurianus) sources; a malic acid synthase gene aceB is knocked out; a malic acid synthase gene glcB is knocked out; and the acetyl phosphate transferase gene eutD is knocked out. The escherichia coli strain which is clear in genetic background and capable of producing gamma-aminobutyric acid is utilized, and an ED path and an acetic acid utilization path are introduced, so that acetyl CoA branch metabolism is reduced, and the yield and conversion rate of gamma-aminobutyric acid are remarkably improved.

The invention discloses a high-concentration malonic acid stress tolerant escherichia coli chassis cell and a construction method thereof. The construction method of the high-concentration malonic acid stress tolerant escherichia coli chassis cell comprises the following steps: carrying out high-concentration malonic acid tolerance domestication on an escherichia coli strain ZXP05 by utilizing an adaptive laboratory evolution (ALE) technology to obtain a high-concentration malonic acid tolerant escherichia coli strain. The strain is subjected to genome re-sequencing, and the result shows that G at the 192nd site from the 5'tail end of the nucleotide sequence of the ompR gene on the genome of the tolerant strain is mutated into C. And mutating G at the 192 site from the 5'tail end of the nucleotide sequence of the ompR gene into C in a homologous recombination manner for a wild escherichia coli strain E.coli C43 (DE3), thereby successfully obtaining the escherichia coli strain W-TB capable of tolerating 400 mM malonic acid. The construction method disclosed by the invention is high in universality, simple and effective, and high osmotic pressure stress of the strain can be relieved; the finally obtained strain W-TB can tolerate high-concentration malonic acid, and the strain can be used as a chassis cell to be applied to biosynthesis of high-added-value compounds by taking high-concentration malonic acid as a substrate and biosynthesis of malonic acid.

The invention provides an Escherichia coli strain for efficiently synthesizing ergothioneine and application of the Escherichia coli strain to fermentation production of ergothioneine. Specifically, the invention provides an escherichia coli strain E1 for expressing high-activity ergothioneine synthetase 1 (cpEGT1-M) and ergothioneine synthetase 2 (clEGT2-M), and 8.6 g / L of ergothioneine is obtained through fermentation. Meanwhile, high-activity S-adenosylmethionine synthetase ctMetK-M is introduced into the strain E1, the synthesis step of S-adenosylmethionine playing a speed limiting role in the strain E1 is accelerated, an escherichia coli strain E2 is obtained, and 15.3 g / L of ergothioneine is obtained through fermentation. Furthermore, high-activity glutathione reductase pfGR-M is introduced into the strain E2, an escherichia coli strain E3 is obtained, and 20.7 g / L of ergothioneine is obtained through fermentation.

The invention provides a genetic engineering strain for efficiently producing beta-alanine, an escherichia coli strain is modified by adopting a genetic engineering means, and a multi-plasmid system is constructed, so that a metabolic pathway is optimized and the product synthesis efficiency is improved. The method comprises the following steps: firstly, by constructing a recombinant plasmid pET-PanD, introducing aspartic acid decarboxylase (L17F / G24R mutant) with mutation so as to promote efficient conversion of aspartic acid to beta-alanine; meanwhile, phosphoenolpyruvate carboxylase (PPC) is overexpressed through the recombinant plasmid pRSF-PPC, generation of oxaloacetic acid (OAA) is increased, and then the synthesis path of carbon flow to beta-alanine is improved. Besides, NADH dependent type aspartate dehydrogenase (AspDH) from pseudomonas aeruginosa is overexpressed by adopting a pBAD-AspDH plasmid, so that the dependence on NADPH is reduced, and the conversion efficiency is further improved.

The invention provides an sgRNA molecule of a targeted IMP-4 gene and a biomacromolecular compound containing the sgRNA molecule, the biomacromolecular compound forms a CRISPR-Cas3 system for targeted removal of the IMP-4 drug-resistant gene, and the invention also provides a method for targeted removal of the IMP-4 drug-resistant gene in a strain by using the system. According to the CRISPR-Cas3 system provided by the invention, the directed gene clearance rate of the strain carrying the IMP-4 drug-resistant gene can reach 100%. The minimum inhibitory concentration of the Escherichia coli subjected to targeted removal of IMP-4 to meropenem is averagely reduced by 71.11 times compared with that of a strain without targeted removal of IMP-4.

The application provides an E. coli outer membrane vesicle targeting TLR2 receptor and a construction method thereof, and relates to the technical field of biological medicine. The application first constructs a ClyA-HLYV fusion protein, and successfully obtains a recombinant plasmid ClyA-HLYV-pGEX-6P-1 stably expressing the fusion protein through genetic engineering technology, and induces endogenous expression of the fusion protein in an E. coli W3110 strain, to prepare a targeted bacterial outer membrane vesicle (HLYV-OMVs) expressing the ClyA-HLYV fusion protein on the surface. The HLYV-OMVs prepared by the application not only retain the specific recognition ability of HLYV to the surface TLR2 receptor of breast cancer cells, but also have the potential to encapsulate drugs, realizing the dual functions of targeted recognition and drug delivery. In addition, the HLYV-OMVs effectively solve the key problem of long-acting time of HLYV single peptide in targeted application, have good stability, significantly accelerate the effect starting speed after HLYV combines with the receptor, and lay an important foundation for efficient targeted treatment of tumors.

The invention relates to application of cyanobacteria acetyltransferase in increasing the content of astaxanthin in escherichia coli for producing astaxanthin, and also provides a method for increasing the content of astaxanthin in escherichia coli for producing astaxanthin, which comprises the step of introducing a cyanobacteria acetyltransferase gene expression cassette into escherichia coli. The method comprises the following steps: amplifying a target gene cKAT from a genome of blue-green algae, inserting the cKAT into an escherichia coli expression vector by using a recombinant DNA technology, and transforming the constructed expression vector into an escherichia coli BW-ASTA strain to obtain recombinant escherichia coli. Therefore, the content of the astaxanthin in the escherichia coli is increased by about 4 times, and a novel efficient, stable and large-scale microbial synthesis scheme is provided for greatly reducing the production cost of the astaxanthin.

The invention discloses an antibiotic-free screening gene expression system, plasmid, engineering bacteria and application thereof, and relates to the technical field of bioengineering. The expression system provided by the invention comprises a first strain, a second strain, a third strain and plasmids of antibiotic-free screening genes, the first strain, the second strain and the third strain are escherichia coli strains with a thyA gene knocked out, the first strain is used for molecular cloning, the second strain is used for expression of target protein, and the third strain is used for biosynthesis of natural products such as polyketone and non-cellulose peptide; a thyA complementary gene expression cassette is inserted into the plasmid of the antibiotic-free screening gene, and a resistance gene is knocked out; the thyA complementary gene expression box contains a thyA gene.

The invention relates to novel application of chlorothiazide, in particular to a bacterial cell membrane bionic nano-drug loaded with an ROS responsive chlorothiazide prodrug and application of the bacterial cell membrane bionic nano-drug. The invention relates to novel application of chlorothiazide and bionic nano-drugs in preparation of drugs for treating inflammatory bowel diseases. The inventor finds for the first time that the Chl active compound can directly drive phenotype reversal of macrophages from M1 to M2 and synchronously repair intestinal mucosal barriers, and breaks the pathogenic closed loop of barrier destruction-inflammation aggravation; however, the colon targeting property is poor, the water solubility is low, and the whole body exposure is limited. Therefore, according to the application, Chl is transformed into an ROS responsive prodrug (CH NPs), and the ROS responsive prodrug (CH NPs) is coated with a cell membrane of an escherichia coli Nissle1917 strain, so that the bionic nanoparticles (CHM NPs) are constructed. The system not only retains the ROS responsiveness of CH NPs, but also obviously improves the targeting ability and residence time of the nanoparticles in a colon area by virtue of the biological characteristics of the bacterial membrane.

The present invention relates to a method for producing a desired linear combined DNA molecule, linear or circular DNA plasmid using at least one genetically altered E. coli strain, wherein linear DNA fragments are converted into a linear combined DNA molecule or circular DNA plasmid. The method can be performed in vivo or in vitro, in particular as a single step method. The invention further relates to a method for cloning a nucleic acid fragment of interest, comprising the method according to the present invention, specific genetically altered E. coli strains, a kit for performing the method according to the present invention, and the use of the genetically altered E. coli strains, or the kit for producing a linear combined DNA molecule or circular DNA plasmid or cloning a nucleic acid fragment of interest.

The present invention relates to a method for high production of heparic acid using a recombinant E. coli strain and to the heparic acid produced thereby. The E. coli strain according to the present invention has the advantage of efficiently increasing the flux of the entire heparic acid biosynthetic pathway through the optimization of the dual plasmid system, promoter combination, and culture medium, thereby increasing the heparic acid production compared to the wild-type strain and enabling the securing of high-purity heparic acid even through a simple purification process.

The invention belongs to the technical field of biology, and relates to an EGFP-LysFEC14CBD fluorescent fusion protein as well as a preparation method and application thereof. In order to solve the technical problem that CBD of target lyase LysFEC14 has potential insolubility, fusion expression is innovatively carried out on the target lyase LysFEC14 and high-solubility EGFP protein, and the EGFP-LysFEC14 CBD fluorescent fusion protein is successfully constructed. The fusion protein disclosed by the invention has remarkable targeting specificity, can recognize various escherichia coli strains from different sources, has extremely low cross reaction to other bacteria (such as vibrio and staphylococcus aureus), and effectively avoids the problem of false positive; the sensitivity is high, the detection limit can be as low as 100 CFU, and the requirements for early warning and detection of trace pollution in a sample can be met; the method can be used for preparing commercial kits, is simple and convenient to operate, is rapid and convenient, and has wide application prospects.

A method for screening and efficiently cultivating bezoar strains belongs to the field of bezoar strain screening, and comprises the following steps: step 1, inoculating a culture medium with low-pathogenicity Escherichia coli for culturing, comparing and observing, picking out single colonies with good growth vigor for subculture, and preserving the strains after culture; 2, taking the primarily screened preserved strain, reviving in four regions of an LB solid culture medium by marking lines, selecting a single colony, putting the single colony into a first gradient liquid bile culture medium, culturing, measuring OD600, and if OD600gt is detected, determining that the single colony is not detected; and 0.6, the strain can be placed in a liquid bile culture medium of the next gradient, and the strain which can grow in the three gradients can be inoculated into fresh oxgall for culture. The method has the advantages that the escherichia coli strain with high beta-glucuronidase activity for efficiently cultivating calculus bovis can be obtained, in-vitro cultivation verifies that the escherichia coli strain has bile resistance, calculus bovis analogues can be obtained, and the physicochemical properties of the calculus bovis analogues are similar to those of natural calculus bovis.

The invention belongs to the technical field of molecular biology, and discloses a chemically competent cell capable of stably storing RecET enzyme as well as a preparation method and application of the chemically competent cell. The technical problem to be solved by the invention is how to solve the problems of high time consumption and low conversion rate of competent cell preparation in DNA cloning. In order to solve the technical problem, the invention provides a composition containing glycerol and calcium chloride and a reagent containing the composition, and the invention also provides a method for stably storing competent cells, and the method comprises the step of mixing the composition or solution with competent cells. And the competent cell can be an escherichia coli GB05-dir strain into which a pSC101-BAD-ETGA-tet plasmid is also introduced.

The invention belongs to the field of biological medicines, and relates to a DDX10 fusion protein and a preparation method thereof. The amino acid sequence of the DDX10 fusion protein is as shown in SEQ ID NO: 1. The method comprises the following steps: 1) amplifying a nucleic acid sequence for coding the DDX10 fusion protein; 2) cloning the nucleic acid sequence into a prokaryotic expression vector; (3) transforming the prokaryotic expression vector into an E. coli strain; 4) inducing expression of the DDX10 fusion protein; and 5) purifying the DDX10 fusion protein. The DDX10 fusion protein disclosed by the invention has ATP hydrolase activity and helicase activity, the method disclosed by the invention is easy for large-scale production and can be used for high-throughput screening of helicase or ATP hydrolase inhibitors, and an expression system of the DDX10 fusion protein is simple.

The present invention relates to probiotic bacteria of the species E. coli, in particular in combination with bacteria of the species Klebsiella oxytoca, that are used for a decolonization of pathogenic and / or multidrug resistant (MDR) Enterobacteria, such as pathogenic E. coli and / or Klebsiella pneumoniae (K. pneumoniae), Enterococci and / or Acinetobacter, from the gut of a subject. The decolonization can both be therapeutic, i.e. after colonization of the gut by the pathogenic and / or multi-resistant pathogen(s), or as a preventive measure before a re-colonization of the gut, as required after antibiotic treatment or treatment-induced dysbiosis.

Provided in the present invention are a manganese ion transporter (MntP) protein mutant and a recombinant strain expressing the mutant for high-yield L-threonine production. The mutant has an amino acid sequence as shown in SEQ ID NO: 2, which is obtained by means of mutating glycine (G) at position 25 to aspartic acid (D) in a wild-type MntP protein sequence as shown in SEQ ID NO: 1. By means of homologous recombination, the mutant is introduced into an Escherichia coli strain, providing the constructed recombinant strain with an increased yield of L-threonine.

This invention discloses a method for targeted screening of teriparatide-producing bacteria using antibiotics. Through two rounds of screening with different concentrations of antibiotics, E. coli strains capable of high teriparatide expression are obtained, and the plasmid stability is effectively maintained during strain passage without the addition of antibiotics. This targeted screening method not only effectively improves the screening efficiency of high teriparatide-producing bacteria but also overcomes the technical difficulty of adding antibiotics during the production of biopharmaceuticals.

The invention belongs to the field of biological medicines, and relates to a DDX54 fusion protein and a preparation method thereof. The amino acid sequence of the DDX54 fusion protein is as shown in SEQ ID NO: 1. The method comprises the following steps: 1) amplifying a nucleic acid sequence of the DDX54 fusion protein; 2) cloning the nucleic acid sequence to a prokaryotic expression vector; (3) transforming the prokaryotic expression vector into an E. coli strain; 4) inducing expression of the DDX54 fusion protein; and 5) purifying the DDX54 fusion protein. The DDX54 fusion protein disclosed by the invention has ATP hydrolase activity and helicase activity, the method disclosed by the invention is easy for large-scale production and can be used for high-throughput screening of DDX54 helicase or ATP hydrolase activity inhibitors, and a protein expression system of the DDX54 fusion protein is simple.

The present invention relates to antibacterial compounds efficient against Escherichia coli, i.e. a composition comprising a cell lysate, wherein the cell produces at least one tailocin from a Pseudomonas bacterium selected from the group consisting of Pseudomonas fluorescens, Pseudomonas chlororaphis, Pseudomonas koreensis and Pseudomonas syringae species, for use in the prevention or the treatment in a subject of a contamination or an infection by an Escherichia coli strain. It further relates to non-therapeutic uses of the same cell lysate for the prevention and / or the treatment of a contamination by an Escherichia coli strain in a food product or on a surface, and intimate care product or a food supplement comprising the cell lysate, as well as new nucleic acids encoding one or two original tailocins, expression vectors or cells comprising them, a cell lysate and its therapeutic and non-therapeutic uses.