21 results about "Coli cell" patented technology

The invention provides a measles virus nucleoprotein specific nano antibody as well as a preparation method and application thereof. The amino acid sequence of the nano antibody is any one of A1-A18; the rash virus nucleoprotein can be specifically recognized and combined. And the nano antibody can be massively expressed and purified in escherichia coli cells, so that a potential nano antibody drug with a great application prospect is provided for clinical prevention, treatment and detection of measles viruses.

The present application relates to the field of microorganisms and genetic engineering, and specifically discloses an Escherichia coli engineering bacterium for efficiently producing plastic depolymerase and application thereof. The present application uses Escherichia coli as a host, and through synergistic optimization of multiple key dimensions such as signal peptide, promoter, solubility-promoting tag and co-expression of molecular chaperones, a multi-dimensional engineering system for efficiently expressing plastic depolymerase is constructed. The construction method provided by the present application includes a rational design and a screening strategy for combination and assembly of the multiple elements. The engineering bacterium can realize extracellular high-level expression of plastic depolymerase during induced fermentation, and the enzyme preparation prepared can be directly used for catalyzing efficient depolymerization of polyester plastics. The present application provides an Escherichia coli cell factory with stable high yield for industrialization and low-cost production of plastic depolymerase, and has important industrial application prospects for promoting large-scale biological degradation and resource recycling of waste plastics.

This invention discloses a directed evolution strategy for SsDMS, a type II sesquiterpene cyclase derived from *Streptomyces showdoensis*, based on alanine scanning, and its applications. The invention describes several key mutant sites in SsDMS capable of catalyzing the generation of novel dried sesquiterpene compounds, including substitutions at positions 208, 248, 249, 497, and 505. This invention also discloses a highly efficient *E. coli* cell factory applied to the heterologous expression of SsDMS mutants and the efficient production of novel dried sesquiterpene compounds, further yielding structurally novel dried sesquiterpene compounds. This invention achieves the efficient production of dried sesquiterpene compounds using SsDMS and its mutants. Its modification strategy has broad applicability, providing important enzymatic resources for the biosynthesis of novel terpenoids and offering more options for the discovery of drug lead compounds.

The invention belongs to the technical field of synthetic biology and biological materials, and particularly relates to a cellulose synthetase high-efficiency expression system based on a cell membrane adaptation strategy, and a preparation method and application of cellulose microfibrils. The method is used for efficient heterologous expression of cellulose synthetase subunits PtCesA1 and PtCesA8 from populus trichocarpa, and in-vivo synthesis of cellulose microfibrils is realized. The key enzyme NMT of a phosphatidylcholine synthesis route is introduced to modify the escherichia coli cell membrane lipid composition, so that the expression efficiency and the cellulose synthesis capability of the eukaryotic-source membrane protein are remarkably improved. Further, the cellulose yield is remarkably increased by systematically optimizing culture conditions such as concentration, temperature and time of the inducer. The system constructed by the invention can be used for directly synthesizing cellulose microfibrils with an ordered structure and high purity in escherichia coli, and a new path is provided for green biological manufacturing of cellulose.

This invention relates to a quantitative method for ultrasonic disruption of *E. coli* cells, specifically a method based on firefly luciferase in *E. coli* cells. The method involves mixing *E. coli* expressing firefly luciferase as an internal standard with a suspension of target protein-expressing bacteria, followed by ultrasonic disruption of the cells. The degree of disruption of the target protein-expressing bacterial suspension is quantitatively calculated by measuring the activity of the firefly luciferase. The target protein-expressing bacterial suspension uses *E. coli* expressing the target protein. Compared with existing technologies, this invention overcomes the shortcomings of complex experiments, low efficiency, and unstable accuracy in existing technologies. It achieves efficient and highly accurate quantification of the degree of ultrasonic disruption of *E. coli* cells, reduces experimental complexity, and improves research and development efficiency and accuracy. This provides a scientific basis and new approach for the development of new feed protein resources and livestock breeding research, and has broad application potential.

The invention relates to the field of whole-cell biosensors, and provides a whole-cell biosensor array and an early warning model for monitoring the mildewing degree and aflatoxin content of corn peanuts. The Escherichia coli cell comprises a promoter related to the response of a volatile organic compound released by Aspergillus flavus pollution, the promoter is operably connected with a reporter gene, and an expression product of the reporter gene is used for generating a bioluminescence signal which can be detected. Through the whole-cell biosensor and the array thereof, related volatile organic compounds after corn peanuts are infected by aspergillus flavus can be accurately responded, so that rapid and high-sensitivity monitoring on aspergillus flavus mildew and toxin pollution is realized. Besides, the whole-cell biosensor array is combined with a machine learning regression model, so that the aspergillus flavus infection degree can be quantified, and the aflatoxin content can be predicted.

The invention discloses a construction method of escherichia coli for efficiently synthesizing glycollic acid, and belongs to the technical field of biochemical engineering. According to the invention, citric acid synthase gltA and aconitic acid synthase acnA and acnB are overexpressed in escherichia coli cells, pyruvate dehydrogenase transcription inhibition factor pdhR is knocked out, transhydrogenase pntA and pntB are overexpressed, and transhydrogenase stA is knocked out, so that the constructed recombinant escherichia coli can produce glycollic acid by using glucose in a short time. The reaction cost is lower, the yield is higher, the concentration of glycollic acid produced after fermentation in a 5L fermentation tank for 48 hours is 81 g / L, the conversion rate is 0.62 g / g, and the production intensity is 1.69 g / L / h.

The invention provides quantum dot modified group B neisseria meningitidis and a preparation method of a vaccine of the quantum dot modified group B neisseria meningitidis. The preparation method comprises the following steps: taking corn straws and sunflower straws as a straw mixture according to a weight ratio of (1-1.5): 1, putting the straw mixture, an escherichia coli DH5alpha cell membrane and urea into water according to a weight ratio of 100: 100: 1, carrying out high-temperature heating reaction, and filtering and dialyzing reactants to prepare aminated quantum dots; extruding the aminated quantum dots and a group B neisseria meningitidis cell membrane through a liposome extruder according to a weight ratio of (8-10): 1, and centrifuging to obtain cell membrane hybrid aminated quantum dots; and co-incubating the cell membrane hybrid aminated quantum dots and the group B neisseria meningitidis to obtain the cell membrane hybrid aminated quantum dots. The group B neisseria meningitidis is modified by adopting the specific quantum dots, the toxicity of the modified group B neisseria meningitidis is obviously reduced, and the modified group B neisseria meningitidis is expected to be applied to related fields of vaccines and the like.

This invention discloses a β-lactoglobulin mutant, its preparation method, and its application, relating to the field of genetic engineering technology. The amino acid sequence of the β-lactoglobulin mutant is shown in SEQ ID NO.1. The preparation method is as follows: designing forward primers, reverse primers, forward mutation primers, and reverse mutation primers; using wild-type β-Lg plasmid as a template, obtaining a mutant DNA fragment using overlap extension PCR technology; ligating the mutant DNA fragment into an empty vector and transforming it into competent E. coli cells for culture and identification to obtain a recombinant plasmid; transforming the recombinant plasmid into competent E. coli cells, culturing, lysing, collecting the cell supernatant, purifying, and obtaining the β-lactoglobulin mutant. This invention, through structural modification of β-lactoglobulin, improves its binding to specific IgE antibodies, enhances the immunogenicity of β-lactoglobulin allergens, and improves the sensitivity and specificity of its allergen-specific IgE antibody detection results.

The application prepares an E.coli-BSO biological composite system for biological hydrogen production. The biological composite system breaks through the limitation of traditional single microorganism and material hydrogen production system, combines bismuth stannate (BSO) nanoparticles and E.coli through electrostatic interaction, and constructs a new composite system with the hydrogen production ability of microbial metabolism and the photocatalytic performance of nanomaterials. The system integrates the inherent hydrogen production metabolic pathway in E.coli cells and the excellent visible light absorption ability and photocatalytic electron transfer performance of BSO nanomaterials, solves the problems of low light energy utilization rate of pure microorganisms and hydrogen production of pure photocatalytic materials depending on external electron donors. Experimental verification shows that under visible light irradiation, the hydrogen production of the composite system reaches 0.8 mmol within 4 hours, which is 1.5 times higher than that of pure E.coli, and significantly breaks through the efficiency bottleneck of existing biological hydrogen production technology.

The invention relates to the technical field of competent escherichia coli cell preparation, and discloses a preparation method of competent escherichia coli cells for gene transformation, which comprises the following steps: step 1, strain activation; step 2, strain amplification culture; step 3, low-temperature suspension and washing: transferring the bacterial liquid into a pre-cooling centrifugal tube, standing on ice, then centrifuging, discarding supernate, adding a pre-cooled CaCl2 solution, gently resuspending, placing on ice, and then centrifuging; step 4, preparation of a competent suspension: removing the supernatant, adding a precooled CaCl2 solution containing glycerol, slightly suspending thalli, and standing on ice for 3-5 minutes to prepare a competent cell suspension; the mass fraction of glycerol in the CaCl2 solution containing glycerol is 15%; the concentration of CaCl2 is 0.05 mol / L; and step 5, sub-packaging and cryopreservation: sub-packaging the obtained competent suspension, and cryopreserving at-80 DEG C. According to the scheme, by optimizing the concentration of CaCl2 and glycerol in the suspension, the suspension temperature and the like, the conversion efficiency, the storage period and the cell membrane stability are effectively improved.

The present disclosure describes a unique viral peptide (VP) vaccine for preventing or treating viral diseases. The vaccine is produced synthetically and includes no production steps in biological cells (e.g. E. coli, CHO cells, yeast cells) that would require subsequent endotoxin assays / removal or viral clearance procedures. The hC peptide is synthesized separately from the VP, and following self-assembly of the hC, the VP is covalently coupled to form the VP-hC conjugate which can serve as a vaccine for preventing or treating viral diseases. The hC includes heptad repeats following a specific pattern. Optionally, the VP-hC conjugate further includes one or more T-cell epitopes at the N- and / or C-terminus of the one or more amphipathic alpha-helices. The present disclosure also describes compositions comprising immunogenic compositions including VP-hC conjugate.

The invention belongs to the technical field of gene engineering, and relates to a method for improving the expression efficiency of penicillin G acylase protein by modifying a signal peptide. According to the invention, E.coli BL21 (DE3) / pET28a-kcPGA is taken as an original strain, an Erwinia carotovora PelB amino terminal leader sequence (the signal peptide is named as pelB) is synthesized through oligonucleotide primer fusion PCR, then the pelB signal peptide is connected with a PGA gene segment without an original signal peptide by using a homologous recombination method, and finally a recombinant plasmid is constructed through seamless cloning, so that the Erwinia carotovora PelB gene fragment is obtained. And the penicillin G is transformed into escherichia coli cells, so that the expression efficiency of the penicillin G acylase is improved. Compared with original recombinant bacteria, the recombinant engineering strain constructed by the invention has the advantages that the protein expression quantity is greatly improved, the enzyme activity is also improved by about two times, the problem of low expression quantity of penicillin G acylase in escherichia coli cells is effectively solved, and the recombinant engineering strain has wider industrial production and application prospects.

The invention discloses a method for screening a melon cold-resistant gene based on a cDNA yeast library, and belongs to the field of gene engineering. The method comprises the following steps: sampling melon leaves subjected to low-temperature treatment, extracting total RNA (Ribonucleic Acid), carrying out reverse transcription to obtain a high-coverage-rate cDNA (Complementary Deoxyribonucleic Acid) library, connecting the cDNA library with a linker to form a three-frame library, connecting the cDNA library to a pYES-NTB carrier, electrically transforming the obtained cDNA library into an escherichia coli competent cell, carrying out cloning amplification, and extracting library plasmids; and transforming the library plasmid and the pYES2 vector into a yeast strain BY4741, screening at low temperature of-20 DEG C for 72 hours, collecting cold-resistant clones, and amplifying and sequencing to obtain the cold-resistant gene of the muskmelon. The constructed yeast system is utilized to efficiently identify potential candidate genes for improving the cold resistance of the muskmelon seedlings, and a foundation is laid for genetic engineering breeding of low-temperature stress resistance of muskmelons.

The invention provides a non-chromatography protein purification label and method suitable for a ship carbon capture biocatalyst-carbonic anhydrase. The method comprises the following steps: connecting a target protein with a purification tag through a proper connecting sequence-linker to construct a target protein-separation tag chimera, constructing the target protein-separation tag chimera in a proper escherichia coli expression vector, introducing the target protein-separation tag chimera into escherichia coli cells, carrying out conventional expansion and induction of strains, collecting bacterial liquid precipitate, and carrying out freeze-drying to obtain the target protein-separation tag chimera. The method comprises the following steps of: taking a crude enzyme as a raw material, resuspending the crude enzyme in a proper buffer solution, crushing at a certain power, collecting crude enzyme precipitate, resuspending the crude enzyme precipitate in the buffer solution, centrifuging at a low speed for 10 minutes, and discarding impurity supernatant, which is a round of purification process, and purifying for 2-3 rounds to obtain the target protein with higher purity.

The invention relates to the field of microorganisms and genetic engineering, and particularly discloses an escherichia coli engineering bacterium for efficiently producing plastic depolymerases and application of the escherichia coli engineering bacterium. According to the invention, escherichia coli is taken as a host, and a multi-dimensional engineering system for efficiently expressing the plastic depolymerases is constructed by synergistically optimizing a plurality of key dimensions such as signal peptide, a promoter, a dissolution promoting tag and a co-expression molecular chaperone. The construction method provided by the invention comprises rational design of the plurality of elements and a screening strategy of combination and assembly. The engineering bacterium can realize extracellular high-level expression of the plastic depolymerization enzyme in the induced fermentation process, and the prepared enzyme preparation can be directly used for catalyzing efficient depolymerization of polyester plastics. The invention provides a stable and high-yield escherichia coli cell factory for industrial and low-cost production of the plastic depolymerases, and has important industrial application prospects for promoting large-scale biodegradation and resource circulation of waste plastics.

This invention relates to the field of biotechnology, specifically disclosing a cell-free protein expression system (CFS), its applications, and a kit. The CFS comprises: 35-50 vol% *E. coli* cell extract, 40-50 vol% lysis buffer, 8-12 vol% gene template, and 2-15 vol% nuclease-free water; wherein the *E. coli* is a recombinant bacterium obtained by modifying a recipient *E. coli*, the modification including: (1) knocking out the LacZ gene in the recipient *E. coli*; and (2) adding the LyseR gene to the recipient *E. coli*. The CFS of this invention features high expression activity and gene stability, making it suitable for expressing high molecular weight proteins and for in vitro diagnostic applications based on cell-free sensors.

This invention discloses an *E. coli* Rosetta strain for the biosynthesis of α-arbutin. Recombinant genes for FruA, CscK, Pgi, and sucrose phosphorylase SmsP are inserted into the genome of the *E. coli* Rosetta strain, anchoring the sucrose phosphorylase SmsP protein product to the surface of *E. coli* cells. The invention also discloses the corresponding transposase plasmids and CRISPR plasmids, as well as the application of the strain in the catalytic synthesis of α-arbutin. This invention utilizes *E. coli* Rosetta (DE3) as the chassis cell and uses CRISPR transposition technology to enhance the FruA, CscK, and Pgi genes in the fructose metabolic pathway, thereby enhancing the metabolic pathway of the reaction's accompanying product, fructose. Enzyme anchoring technology is used to anchor sucrose phosphorylase to the surface of *E. coli*, allowing the sucrose phosphorylase to grow on the *E. coli* surface and catalyze the conversion of sucrose and hydroquinone from the external environment into α-arbutin and fructose.

The embodiment of the present application discloses a method for identifying polypeptide antigen epitopes and application. The method for identifying polypeptide antigen epitopes comprises the following steps: transforming E. coli cells with an expression vector containing nucleic acid encoding a target protein, and culturing the E. coli cells; wherein the target protein is expressed in the form of a fusion protein, the fusion protein comprising: a polypeptide to be identified; an S tag at the N terminus thereof; and a targeting sequence at the C terminus thereof, the targeting sequence targeting the fusion protein to the extracellular domain of the E. coli cells. The E. coli supernatant containing the fusion protein is combined with a solid-phase medium, immunological color development and detection of antibody specificity are performed. The method provided by the present application can efficiently express and secrete target polypeptides, and can directly use the culture medium for epitope identification of antibodies.

The subject of this invention is a construct of an expression vector for expressing recombinant µ-conotoxin TIIIA or TIIIAlaMut, characterized in that it comprises the nucleotide sequence of µ-conotoxin TIIIA SEQ ID NO:1 or the nucleotide sequence of µ-conotoxin TIIIAlaMut SEQ ID NO:4, both sequences containing a sequence encoding six histidine residues (6His) at the 5' end, which is linked via a serine-glycine-serine linker (SGS) to a construct encoding a TRX::TIIIA fusion protein or a TRX::TIIIAlaMut fusion protein, wherein the TRX::TIIIA fusion protein comprises the µ-conotoxin TIIIA gene and a gene encoding a leader protein, and the TRX::TIIIAlaMut fusion protein comprises the µ-conotoxin TIIIAlaMut gene and a leader protein, wherein the leader protein is a thioredoxin (TRX) modified by site-directed mutagenesis, wherein the amino acid methionine at position 37 is replaced by lysine. Another subject of the invention is an expression vector comprising a construct according to the invention under the control of a constitutive promoter. Another subject of the invention is isolated *E. coli* cells comprising an expression vector according to the invention. Another subject of the invention is a method for producing µ-conotoxin TIIIA or TIIIAlaMut in *E. coli* using an expression vector comprising a construct according to the invention, characterized in that the method comprises the steps of: a) transforming *E. coli* cells with an expression vector comprising a construct according to the invention under the control of a constitutive promoter, said expression vector encoding a TRX::TIIIA fusion protein having the amino acid sequence SEQ ID NO:2 or having SEQ ID NO:2. a) TRX::TIIIAlaMut fusion protein NO:5; b) Expression of the TRX::TIIIA or TRX::TIIIAlaMut fusion protein; c) Isolation and purification of the TRX::TIIIA or TRX::TIIIAlaMut fusion protein; d) Formation of disulfide bonds by glutathione treatment of the purified TRX::TIIIA or TRX::TIIIAlaMut fusion protein in GSH / GSSG and dialyzing in buffer; e) Cleavage of the TRX::TIIIA or TRX::TIIIAlaMut fusion protein with the formed disulfide bonds by cyanogen bromide; f) Purification of the cleaved TIIIA or TIIIAlaMut peptide.

The application discloses construction and application of multiple electron transfer pathway Escherichia coli, selects non-natural electrically active microorganism Escherichia coli as a research object, and introduces the electron transfer pathway of natural electrically active microorganism into the Escherichia coli. In the Escherichia coli with the Mtr pathway, the appropriate endogenous electron mediator is introduced, and the multiple electron transfer pathways are used to strengthen the transmembrane electron transfer of the Escherichia coli. In order to further improve the electron transfer capacity, the IPTG concentration, the induction temperature, the induction time, the induction initial OD are optimized, the best fermentation condition is selected, the production of phenazine-1-carboxylic acid is improved, and the electron transfer efficiency is improved.