135 results about "Bacterium coli" patented technology

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 belongs to the technical field of biosynthesis, and particularly relates to a strain for producing sclareol as well as a construction method and application of the strain. In order to solve the problem of microbial synthesis of sclareol, phosphoketolase in an NOG pathway is mutated and optimized, phosphoketolase NaXpk from nanoarchaea archaeon is subjected to site-directed mutagenesis, a sclareol synthesis pathway is constructed in an escherichia coli host, the influence of NaXpk before and after mutation on the yield of sclareol is compared, and the yield of sclareol is improved. The S472F mutant and the S472Y mutant are determined, the S472F mutant and the S472Y mutant are applied to a sclareol production path, the sclareol yield of the finally obtained production strains can reach 623.3 mg / L and 655.2 mg / L, and the effect is remarkable.

The invention discloses a method for improving the solubility and thermal stability of sweet protein, and belongs to the technical field of biosynthesis, the method comprises the following steps: constructing a gene tandem recombinant plasmid containing a monellin x-3C-sfGFP-3C-monellin y expression cassette, x is greater than or equal to 1, y is greater than or equal to 1, 5 is greater than or equal to x + y is greater than or equal to 3, a 3C protease recognition sequence is also inserted between adjacent copies of monellin, and each monellin is connected with a purification tag; the gene tandem recombinant plasmid is transformed into escherichia coli and inducible expression is carried out, thalli are split and purified to obtain fusion protein, 3C protease is used for enzyme digestion, and the target protein is obtained after re-purification. The total protein yield and the solubility proportion are remarkably improved by adopting a series construction mode of plasmids, meanwhile, the stability of monellin is effectively improved, the purification step is simpler and more convenient, and the method is suitable for industrial production.

The invention belongs to the technical field of synthetic biology genome editing, and relates to a chassis strain for gene cluster in-vivo traceless editing, a construction method and a gene cluster traceless editing method.The chassis strain is based on escherichia coli BL21 (DE3) and integrates a CRISPR-Cas9 system and a homologous recombination system (lambda Red) of a lambda bacteriophage source; and then knocking in an inducible sgRNA expression cassette (sgT) of a targeted editing vector to construct the vector. The strain has an efficient editing capability aiming at exogenous DNA, traceless editing of a target biosynthetic gene cluster is realized, and editing plasmids can be quickly eliminated, so that iterative editing is facilitated. The method is simple, convenient and rapid to operate and high in practicability, and provides powerful technical support for metabolic engineering and synthetic biology breeding transformation of streptomyces industrial strains, activation of synthetic gene clusters of recessive natural products and excavation of novel natural products.

Methods of producing bioconjugates of O-antigen polysaccharides covalently linked to a carrier protein using recombinant host cells are provided. The recombinant host cells used in the methods described herein encode a particular oligosaccharyl transferase enzyme depending on the O-antigen polysaccharide bioconjugate to be produced. The oligosaccharyl transferase enzymes can be PglB oligosaccharyl transferase or variants thereof. Also provided are compositions containing the bioconjugates, and methods of using the bioconjugates and compositions described herein to vaccinate a subject against extra-intestinal pathogenic E. coli. (ExPEC).

The invention provides a complex microbial inoculant for producing beta-glucuronidase and application of the complex microbial inoculant in calculus bovis production, and relates to the technical field of microorganisms. The culture method of the complex microbial inoculant provided by the invention comprises the following steps: culturing lactobacillus ATCC 53103 in an initial culture medium to a logarithmic phase, sterilizing to obtain a complex culture medium, mixing the complex culture medium with nutritional ingredients, inoculating escherichia coli ATCC 25922 and riemerella CCUG 14215, and co-culturing to obtain the complex microbial inoculant. The complex microbial inoculant provided by the invention can generate a large amount of high-activity beta-glucuronidase, so that the efficiency of in-vitro production of calculus bovis can be effectively improved.

The invention discloses a genetically engineered bacterium for biosynthesis of gastrodin as well as a construction method and application of the genetically engineered bacterium. According to the genetically engineered bacterium, through overexpression of sucrose permease CscB derived from escherichia coli W, sucrose phosphorylase Basp of bifidobacterium adolescentis, uridine triphosphoryl-glucose-1-phosphate uridine acyl transferase UgpA of bifidobacterium bifidum and UDP-glycosyl transferase RsUGT of hedyotis diffusa, an exogenous path for synthesizing UDP-glucose from sucrose is constructed in a cell, and the UDP-glucose is synthesized into UDP-glucose. And the glycosylation capability of the gastrodin precursor on hydroxybenzyl alcohol is enhanced. The invention also provides a construction method of the genetically engineered bacterium and a method for synthesizing gastrodin through whole-cell catalysis by using the strain and taking p-hydroxybenzyl alcohol and cane sugar as substrates. The genetically engineered bacterium is high in gastrodin synthesis efficiency, the molar conversion rate can reach 97.8%, the highest yield reaches 3.61 g / L, and a new green manufacturing scheme is provided for solving the problems that a traditional gastrodin production method is low in efficiency, high in cost, large in pollution and the like.

The invention provides a method for producing beta-glucuronidase from escherichia coli and application of the beta-glucuronidase, and relates to the technical field of microorganisms. The method provided by the invention comprises the following steps: inoculating Escherichia coli ATCC 25922 on a composite culture medium for pre-culture, and culturing in an electric field environment of 1-10N / C for 12-24h to obtain a mixture containing beta-glucuronidase; wherein the composite culture medium comprises 10-100 [mu] mol / L of ferric citrate, and the concentration of the ferric citrate is increased in stages in the pre-culture process. According to the method, the escherichia coli ATCC 25922 is cultured in the iron-containing culture medium and the low-intensity electric field environment, so that the synthesis of the beta-glucuronidase by the escherichia coli ATCC 25922 can be effectively improved, and the enzyme activity and the enzyme yield of the beta-glucuronidase are improved.

The subject relates to an isolated antibody that specifically binds to O25b antigen of multi drug resistant (MDR) E. coli strains, its medical and diagnostic use, method of producing the antibody, including an isolated nucleotide sequence, plasmids and host cells as used in the production of the antibody; and further an isolated epitope recognized the specific antibody.

The invention provides a quality control kit of a nucleic acid detection system as well as a quality control method and application of the quality control kit. The quality control kit comprises the coliphage MS2 and a first sequence group. The first sequence group comprises a forward primer of a sequence as shown in SEQ IQ NO: 1, a reverse primer of a sequence as shown in SEQ IQ NO: 2 and a probe of a sequence as shown in SEQ IQ NO: 3. The quality control method comprises the following steps: mixing a specimen with bacteriophage MS2, and carrying out nucleic acid extraction to obtain an eluent; and mixing an eluent with the feline blood plasma bacterial plastid, the first sequence group and the second sequence group, and carrying out multiple real-time polymerase chain reaction. The quality control kit of the nucleic acid detection system is used for evaluating the recovery rate of nucleic acid and the whole-process quality control of the nucleic acid detection system.

The invention provides an antibacterial agent based on co-assembly of an anionic surfactant and polyarginine, and particularly relates to a safe and efficient antibacterial agent. The antibacterial agent provided by the invention is an aggregate formed by co-assembly of polyarginine (Arg) n and an anionic surfactant, and the preparation method is simple. The antibacterial agent is high in safety and has no toxicity to mammalian cells; the compound has remarkable antibacterial activity on escherichia coli and staphylococcus aureus. According to the invention, the assembly structure of the antibacterial agent can be regulated and controlled by adjusting the number (such as 4, 8, 12 or 16) of arginine in the polyarginine and the charge ratio between the polyarginine and the anionic surfactant; and when the layered structure is formed, the antibacterial agent has the strongest antibacterial activity. Meanwhile, in a mouse in-vivo experiment, the antibacterial agent also shows efficient antibacterial activity and can promote wound healing.

The invention relates to the technical field of aquatic animal immune regulation and control, in particular to rHoLL-like stepped lectin-like recombinant protein as well as a preparation method and application thereof. The recombinant protein has pathogen-related molecular pattern recognition and combination capabilities, can promote aggregation of pathogens in a body fluid environment, and activates an aquatic animal complement system, so that the non-specific immune defense capability is enhanced. The preparation method comprises the following steps: amplifying a HoLL-like mature peptide coding sequence, constructing a recombinant expression vector, and converting the recombinant expression vector into an escherichia coli expression host for induced expression to obtain rHoLL-like lectin-like recombinant protein existing in an inclusion body form; and carrying out affinity chromatography purification on the inclusion body protein, and carrying out renaturation treatment in a manner of gradient reduction of denaturant concentration to obtain the rHoLL-like stepped lectin-like recombinant protein. The rHoLL-like stepped lectin-like recombinant protein has good biological safety, and can be applied to aquaculture as an aquatic animal immunopotentiator or a related biological product.

The application discloses a Mycoplasma bovum adhesion protein FtsZ and application thereof. The nucleic acid sequence of the Mycoplasma bovum adhesion protein FtsZ is shown as SEQ ID NO. 1. The application also discloses a recombinant plasmid pET-30a-ftsZ and an E. coli containing the recombinant plasmid pET-30a-ftsZ. ftsZ The recombinant protein rFtsZ has the advantages of being capable of specifically combining with EBL cell membrane protein, being capable of combining with extracellular matrix components (fibronectin, fibronectin, laminin and type IV collagen), having the direct adhesion host cell effect, having good antigenicity, being capable of producing high-level antibodies, being capable of providing good immune protection effect, and providing a new target and thought for elucidating the pathogenic mechanism of the Mycoplasma bovum and developing a new vaccine and medicine.

The invention relates to the field of gene editing, in particular to a gene editing system based on Escherichia coli endogenous EcoTnpB protein and application of the gene editing system, and the gene editing system comprises a) EcoTnpB protein expressed by Escherichia coli endogenous or a nucleotide sequence for coding the EcoTnpB protein; and b) an omega RNA molecule or a nucleotide sequence encoding the omega RNA molecule, the sequence of the omega RNA molecule comprises an RNA scaffold sequence and a guide sequence complementarily paired with a target sequence, and the omega RNA molecule can form a complex with the EcoTnpB protein and guide the EcoTnpB protein to recognize the target sequence. Escherichia coli endogenous protein is combined with customized omega RNA and a corresponding editing template for genome editing, efficient knockout, insertion and continuous editing of target genes can be achieved in Escherichia coli, and the method has wide application value in different Escherichia coli hosts.

The invention discloses an antibiotic resistance prediction method based on a metric element learning architecture. According to the framework, an adaptive mechanism is adopted, and the characteristics of different antibiotics and the distribution characteristics of genome data are precisely matched with an adaptive machine learning model by deeply analyzing the characteristics of the different antibiotics and the distribution characteristics of the genome data. Escherichia coli whole genome sequencing data is adopted, and prediction research is carried out aiming at whether antibiotics have drug resistance or not, so that an optimal prediction model under different drug types and data distribution conditions is evaluated. In addition, molecular structure characteristics and data distribution modes of antibiotics are deeply excavated, and a metrics-based meta learning model matching mechanism is constructed. According to the method, through a metrics-based meta learning framework, the problem of model selection in antibiotic resistance prediction in a complex scene can be effectively solved; meanwhile, in a new drug resistance prediction task, a zero sample learning ability is realized, model adaptation can be completed without extra training data, and the computing resource consumption cost in a model training process is effectively reduced.

The application discloses a method for constructing a tag sequence-containing vector library by short oligonucleotides and application thereof. The method needs two oligonucleotides, i.e. a customized tag primer and a universal primer which can be used for all library constructions. The middle of the tag primer is a tag sequence, and both sides are annealing sequences. The universal primer has sequences complementary to the annealing sequences of the tag primer on both sides, and a modified base which can be recognized as a base damage by a host microorganism in the middle. The tag primer library is annealed with an equal amount of the universal primer to obtain primer dimers. The primer dimers have base-paired DNA double strands on both sides, and are cohesive ends. The middle of the primer dimers is an unpaired omega loop structure, and the double strands of the omega loop contain the tag information carried by the tag primer and the modified base carried by the universal primer respectively. The primer dimers are connected with linearized vectors, and then are transformed into E. coli, so that the region of the modified base of the omega loop is replaced by the tag sequence through the base excision repair mechanism of the endogenous cells.

The application belongs to the field of enzyme engineering and biotechnology, and discloses a 4-hydroxybenzoic acid hydroxylase mutant and application thereof in synthesis of gallic acid or a salt thereof. The 4-hydroxybenzoic acid hydroxylase mutant is obtained through directional evolution modification and high-throughput screening, and has an increased protocatechuate hydroxylase activity by more than 4 times. The 4-hydroxybenzoic acid hydroxylase mutant is expressed by using E. coli, and the obtained recombinant E. coli can efficiently catalyze the conversion of protocatechuate into gallic acid. The 4-hydroxybenzoic acid hydroxylase mutant is expressed in the recombinant E. coli for producing protocatechuate, and the obtained recombinant E. coli can de novo synthesize gallic acid from glucose as a substrate, and the yield reaches 58.65 g / L, and the residual amount of protocatechuate is only 0.33 g / L. The application solves the problem of a large amount of residual protocatechuate in the fermentation process of the existing strains, and can provide an efficient enzyme element for the creation of gallic acid industrial strains.

The invention provides a plasmid and application thereof. The plasmid comprises a recognition site of a first endonuclease, a target gene and a recognition site of a second endonuclease from a 5 '-3' direction. According to the invention, the plasmid is amplified by using a rolling circle amplification method, the rolling circle amplification product is cleaved by using the first endonuclease and the second endonuclease, the enzyme cleaved product is digested by using exonuclease, and the product is further purified by using oligonucleotide. The obtained product can be used for in vitro transcription or directly applied to eukaryotic expression. According to the scheme provided by the invention, a simple, high-yield, economic, rapid and amplifiable preparation process of the full chemical synthesis DNA template for escherichia coli fermentation is established.

The present invention relates to a method for treatment of polyhydroxyalkanoate (PHA) containing post-consumer product, the method comprising contacting a post-consumer product with a polypeptide that can catalyze degradation of the PHA, the contact taking place at a temperature at least 45° C. In a specific embodiment, the poly peptide is a wild-type PHA depolymerase expressed by a thermophilic microorganism or a modified PHA depolymerase that includes one or more single-site mutations as compared to the wild-type PHA depolymerase. In another specific embodiment, the polypeptide comprising a modified poly hydroxy butyrate (PHB) depolymerase comprising one or more single-site mutations as compared to SEQ ID NO: 1, and the modified PHB depolymerase having an optimum temperature of at least 45° C. The present invention also relates to a host cell transformed to express a polypeptide that catalyzes degradation of a PHA, the polypeptide having an optimum temperature for the degradation reaction of at least 45° C., wherein the host cell is selected from an E. coli cell or a thermophilic microorganism.

The present invention relates to a method for treatment of poly hydroxy alkanoate (PHA) containing post-consumer product, the method comprising contacting a post-consumer product with a polypeptide that can catalyze degradation of the PHA, the contact taking place at a temperature at least 40° C. and in the presence of salt at a concentration of 1 M or greater. In a specific embodiment, the polypeptide is a wild-type PHA depolymerase expressed by a halophilic microorganism or a modified PHA depolymerase that includes one or more single-site mutations as compared to the wild-type PHA depolymerase. In another specific embodiment, the polypeptide comprising a modified poly hydroxy butyrate (PHB) depolymerase comprising one or more single-site mutations as compared to SEQ ID NO: 1, and the modified PHB depolymerase having a solubility of 10 mg / L or greater. The present invention also relates to a host cell transformed to express a polypeptide that catalyzes degradation of a PHA in the presence of salt at a concentration of 1 M or greater, wherein the host cell is selected from an E. coli cell or a halophilic microorganism.

The invention relates to the technical field of biology, and particularly discloses a construction method and application of escherichia coli engineering bacteria for overexpressing uric acid degradation gene clusters. According to the invention, a mixed promoter box is inserted into Escherichia coli, overexpression coding is carried out on genes of regulatory protein YgeV and key genes in a uric acid degradation pathway, and the Escherichia coli engineering bacteria which can overcome inhibition of intestinal glucose on uric acid degradation and enable the Escherichia coli engineering bacteria to overexpress uric acid degradation gene clusters are successfully constructed. The coliform engineering provided by the invention provides a new strategy for prevention and treatment of hyperuricemia and gout.

The invention provides a mutant of a YciE gene coding protein and application of the mutant in preparation of NMN, belongs to the technical field of molecular biology, and particularly provides the mutant of the YciE gene coding protein, the amino acid mutation site is that the 140th methionine of the amino acid sequence SEQ ID NO.3 of the YciE gene coding protein is mutated into isoleucine; the coding gene of the mutant is as shown in SEQ ID NO. 2; a recombinant bacterium containing the coding gene of the mutant; according to the invention, it is found for the first time that methionine at the 140th site in a protein amino acid sequence coded by a YciE gene of escherichia coli is mutated into isoleucine; the synthesis level of NMN can be effectively improved, and generation of by-products is reduced.

Disclosed herein are prokaryotic and eukaryotic microbes, including E. coli and S. cerevisiae, genetically altered to biosynthesize tryptamine and tryptamine derivatives. The microbes of the disclosure may be engineered to contain plasmids and stable gene integrations containing sufficient genetic information for conversion of an anthranilate or an indole to a tryptamine. The fermentative production of substituted tryptamines in a whole-cell biocatalyst may be useful for cost effective production of these compounds for therapeutic use.

The present description relates to a recombinant microorganism having production activity of pentamethylenediamine (PMDA), which comprises an E. coli Nissle-derived decarboxylase enzyme protein, and uses thereof.

The invention is applicable to the field of gene engineering, and provides a mycobacterium tuberculosis Ag85B-TB8.4-LS protein nanoparticle, a preparation method and application thereof, the mycobacterium tuberculosis Ag85B-TB8.4-LS protein nanoparticle is formed by sequence fusion of mycobacterium tuberculosis Ag85B protein, TB8.4 protein and LS protein, and the amino acid sequence of the mycobacterium tuberculosis Ag85B-TB8.4-LS protein nanoparticle is shown as SEQ ID NO: 2 in a sequence table. According to the invention, by virtue of a gene recombination technology, key immunogens Ag85B and TB8.4 of mycobacterium tuberculosis are combined with an LS protein fusion vector, and the Ag85B-TB8.4-LS protein nanoparticles with uniform particle size and stable structure are efficiently produced by virtue of an escherichia coli expression system. Animal experiment results show that the Ag85B-TB8.4-LS protein nanoparticle can simultaneously excite strong humoral immunity and cellular immunity response, not only brings a new idea for research and development of tuberculosis vaccines, but also can be popularized and applied to research and development of other infectious disease vaccines due to the modular design, and has important scientific significance and industrialization prospects.

The application discloses a human source pioneer transcription factor SOX15 HMG domain recombinant protein and a preparation method thereof, belongs to the technical field of biology, and specifically comprises the following steps: optimizing the expression SOX15 HMG protein condition of E. coli, purifying the SOX15 HMG protein, and detecting the activity of the SOX15 HMG protein after purification. It is found through the exploration of the optimized expression condition that the soluble protein can be obtained by using 0.1-0.5 mM of the concentration of an inducer IPTG (isopropyl-beta-D-thiogalactoside) and inducing for 3-4 hours at 37 DEG C. The two-step purification strategy of nickel column affinity chromatography combined with molecular sieve chromatography can obtain the recombinant protein with a purity of more than 90% and a molecular weight of about 12 kDa. Electrophoretic mobility shift assay (EMSA) proves that the purified SOX15 HMG recombinant protein has the activity of binding to target DNA.

The invention relates to the field of microbiology, specifically to an antimicrobial composition comprising a first and a second bacteriophage, wherein the composition has lytic activity against E. coli O157. The invention further relates to a use of the antimicrobial composition for controlling bacterial contamination in a food- or feed environment on or in food- or feed processing equipment or food- or feed containers or in a food- or feed product.

The present invention provides a fluorinase mutant and a microbial cell displaying fluorinase on the cell surface and its application, belonging to the field of genetic engineering technology. In order to solve the problem of substrate transmembrane restriction in the whole cell catalysis process. The present invention provides a gene with a sequence as shown in SEQ ID NO: 3, and the Escherichia coli engineering bacteria is an Escherichia coli engineering bacteria obtained by using Escherichia coli as a starting strain and expressing an ice nuclease N-terminal encoding gene and a fluorinase encoding gene. The engineering bacteria can stably express the fluorinase gene in Escherichia coli and automatically display it on the cell surface, directly contacting the enzyme with the substrate, avoiding the restriction of the substrate passing through the cell membrane, maximizing the utilization rate of the enzyme, and reducing the influence of exogenous proteins on the host by surface display. A larger cell amount can be obtained, and the possibility of engineering application is improved.

The present specification relates to a recombinant microorganism comprising an E. coli Nissle-derived decarboxylase enzyme protein and having pentamethylenediamine (PMDA)-producing activity, and a use thereof.

The invention discloses a nucleotide sequence of PDRN / PN and a preparation method of PDRN / PN based on genetic engineering and microbial fermentation, and belongs to the technical field of biology. According to the method, the vibrio is adopted as a production chassis strain, and methods of pyrolysis crude extraction, enzyme digestion and magnetic bead selective separation are combined, so that the preparation of the multi-source PDRN / PN is realized, and the application range of the product is expanded. Compared with an escherichia coli system, the method has the advantages that the endotoxin pollution risk can be remarkably reduced, and the product safety is improved; compared with a yeast system, the production efficiency of PDRN / PN can be improved. Meanwhile, the method is simple and convenient to operate, is suitable for laboratory and pilot scale amplification, and has obvious industrial application potential.