54 results about "Coli cell" patented technology

The present invention relates to a recombinant human type IV collagen and a preparation method therefor, which belong to the technical field of bioengineering. A recombinant human type IV collagen is designed on the basis of a partial sequence in an α2 chain of human type-IV collagen, and DNA encoding of the protein is ligated into an expression vector to construct a recombinant expression vector; then the recombinant expression vector is transformed into competent Escherichia coli cells, and a recombinant expression plasmid is extracted; after the plasmid is linearized, the linearized plasmid is transformed into an engineered strain HCPB-PPKEX2 to obtain a recombinant engineered strain; and the engineered strain is cultured, the expression of a target protein is induced, and the target protein is purified and identified. It is further demonstrated experimentally that the collagen can be completely cleaved by Kex2, Ste13 and CPB enzymes and efficiently secreted and expressed extracellularly, and the obtained sequence is completely consistent with a theoretical sequence. It is also verified experimentally that the recombinant collagen has a cell adhesion activity and a cell migration-promoting activity. The method avoids the risk of residual exogenous proteins, shortens the time and reduces the cost of the subsequent purification process, and thus has a relatively high safety and application value.

The invention belongs to the technical field of bioengineering, and discloses a plasma membrane localized green fluorescence overexpression vector as well as a construction method and application thereof. The cytoplasmic membrane localized green fluorescence overexpression vector contains an expression vector skeleton and an AtCBL1 gene connected to the expression vector skeleton, wherein the nucleotide sequence of the AtCBL1 gene is shown as SEQIDNO: 1, and the expression vector skeleton also carries a fluorescent protein gene. The construction method of the vector comprises the following steps: S1, carrying out enzyme digestion and recovery on an AtCBL1 gene sequence with a nucleotide sequence as shown in SEQIDNO: 1 obtained by amplification; s2, performing same enzyme digestion on the expression vector carrying the fluorescent protein gene, and recovering; and S3, connecting the AtCBL1 gene subjected to enzyme digestion with the expression vector to form a recombinant plasmid, converting a connection product into an escherichia coli competent cell, and extracting the recombinant plasmid from positive clone to obtain the plasma membrane localized green fluorescence overexpression vector.

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 discloses a preparation method of glutamate oxidase and its related applications, which relates to the field of genetic engineering. The present invention utilizes the mcherry tag to promote the display of glutamate oxidase on the surface of Escherichia coli cells. Through the solubilization promotion of the tag, cells that can catalyze the substrate L-glutamate to prepare α-ketoglutaric acid are obtained, providing a new idea for the industrial production of α-ketoglutaric acid and also providing a new way for the heterologous expression of L-amino acid oxidase.

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.

The invention relates to the technical field of production of cholyl glycine hydrolase, and discloses recombinant escherichia coli for expressing the cholyl glycine hydrolase and application of the recombinant escherichia coli. According to the invention, CGH derived from Bifidobacterium breve is subjected to preference codon optimization, and is introduced into Escherichia coli to obtain recombinant Escherichia coli for fermentation, and high-activity CGH is separated from thalli of the recombinant Escherichia coli. Specifically, the yield of CGH expressed by the gene reaches 116 mg / L, and the activity of the prepared CGH reaches 664 U / mg. Therefore, the problem of low activity of CGH obtained by existing escherichia coli expression is solved. The amino acid sequence of the CGH derived from bifidobacterium breve is as shown in SEQ ID No. 1, and the nucleotide sequence of the CGH derived from bifidobacterium breve is as shown in SEQ ID No. 2.

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 provides a method for extracting and purifying CRM197 protein, and belongs to the technical field of vaccine preparation. Specifically, CRM197 protein is expressed in escherichia coli cytoplasm by adopting a method without adding signal peptide and fusion tag, thalli are treated by an osmotic pressure method (repeated freezing and thawing + hypertonic solution + hypotonic solution) so as to extract soluble expression CRM197 protein in cytoplasm, and then two-step column chromatography (anion column chromatography + hydrophobic column chromatography) is carried out to obtain the CRM197 protein in the cytoplasm. The target protein with relatively high purity is obtained. The CRM197 protein extraction and purification method provided by the invention is simple, efficient and easy to operate, does not need the assistance of complex equipment, and has a cost advantage.

The invention belongs to the technical field of synthetic biology, and particularly relates to a multi-host in-vivo continuous evolution system and method and application thereof. Specifically, a target plasmid containing a target gene and an OriV replicon is targeted through combined expression of an initiation protein RepC in a broad-host plasmid RSF1010 replication system, TadA8e-RepA fused with deaminase TadA8e and helicase RepA, and RepB-PolI auxiliary plasmids fused with an initiation enzyme RepB of error-prone PolI, and the invention provides an effective in-vivo continuous evolution method. The method not only can be evolved in escherichia coli cells, but also can be used for other bacteria and fungi, so that the method has considerable application value and prospect.

The invention provides a nano antibody targeting coronavirus RBD protein and application thereof, and relates to the technical field of biological medicine, and the amino acid sequence of the nano antibody is any one of A1 to A4. According to the present invention, the affinity between the nano antibody and the SARS-CoV-2S protein receptor binding region is high, the half inhibitory concentration of the SARS-CoV-2 pseudovirus and true virus infection inhibition achieves 20-100 nM, the Delta variant infection can be effectively inhibited, and the good broad-spectrum neutralization effect is provided; a large amount of the gene can be expressed and purified in escherichia coli cells, and the purity is very high; a single nano antibody is subjected to tandem expression by using a G4S linker, so that higher neutralizing activity and wider-spectrum neutralizing capacity are shown. The invention provides a potential nano antibody drug with a great application prospect for clinical prevention, treatment and detection of the SARS-CoV-2 original strain and the variant strain thereof.

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.

The invention relates to the technical field of biology, in particular to a Cas protein mutant, a fusion protein and application of the Cas protein mutant and the fusion protein in gene editing. The research finds that the Cas12h protease complex has the capability of specifically cutting a non-target chain (NTS) in double-stranded DNA in vitro, so that the open loop of the DNA double-stranded DNA is triggered. Based on the characteristic, the invention further develops a gene silencing function of Cas12h in vivo, and successfully realizes a silencing effect on transcription and expression of a specific gene in escherichia coli. Meanwhile, an adenine base editor based on Cas12h fusion protein is further developed, and accurate base conversion from A-T to G-C on the DNA level is successfully realized in escherichia coli cells by finely regulating and controlling growth conditions of the base editor, optimizing peptide connector design and reasonably constructing a fusion protein structure.

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 application discloses a kind of recombinant escherichia coli for producing NMN and its preparation method and application, belong to genetic engineering and bioengineering technical field.The application expresses nicotinamide phosphoric acid ribosyltransferase NAMPT as shown in SEQ ID NO.1 in escherichia coli, expresses PRPP synthetase BaPRS as shown in SEQ ID NO.2, expresses transporter BMpnuC as shown in SEQ ID NO.3, and knocks out pncC gene, ushA gene, nadR gene and purR gene, improves the accumulation amount of NMN in escherichia coli cell.The application also optimizes fermentation condition, so that NMN can be accumulated efficiently outside cell, and more than 20g / L NMN can be obtained in 5L fermentation system, and has wide application prospect in the field of medicine, cosmetics, feed, textile and the like.

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 application provides a method for extracting and purifying CRM197 protein, and belongs to the technical field of vaccine preparation. Specifically, the CRM197 protein is expressed in the cytoplasm of Escherichia coli by a method without signal peptide and fusion tag addition, and the bacterial cells are treated by an osmotic pressure method (repeated freeze-thawing + high-osmotic solution + low-osmotic solution) to extract the CRM197 protein expressed in the cytoplasm, and then two-step column chromatography (anion column chromatography + hydrophobic column chromatography) is performed to obtain the target protein with high purity. The method for extracting and purifying the CRM197 protein is simple, efficient, easy to operate, does not need complex equipment assistance, and has a cost advantage.

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.

The invention discloses an electrochemical luminescence sensor based on bacterial surface display recombinant fusion protein. The recombinant fusion protein comprises an ice nucleation protein N-terminal structural domain and eel fluorescent protein. According to the invention, an ice nucleation protein N-terminal structural domain is used as a bacterial surface anchoring element, so that the UnaG protein is actively expressed on the bacterial surface through a surface display system, and the problem that in the prior art, the expression of an exogenous protein in escherichia coli cells needs to involve complicated subsequent steps such as bacterium breaking and purification is solved; and the effect of a biosensor is achieved by utilizing the electrochemical luminescence characteristic of the compound after the compound is combined with bilirubin, so that an efficient and sensitive detection means is brought to the field of biosensing.

This invention relates to the field of genetic engineering technology, and particularly to TEV protease mutants, their preparation methods, and applications. This invention modifies the TEV enzyme molecule, with mutation points differing from those reported in previous studies. This enhances the activity of the TEV protease at the molecular level. Furthermore, this invention utilizes CRISPR / Cas9 gene editing techniques to modify the host bacterial chassis, obtaining engineered strains that express TEV at high yields. Further, the provided high-density fermentation control method can rapidly promote TEV enzyme expression, resulting in high E. coli cell yield, with an OD600 value reaching approximately 150, a cell wet weight of 80–130 g / L, and high expression levels of the target protein, accounting for 40%–64% of the total bacterial protein. Simultaneously, the method provided by this invention is simple and easy to implement, using readily available raw materials, achieving good application results while reducing production costs, resulting in significant economic benefits.

The invention relates to an Escherichia coli (E. coli-BSO) biological compound system for biological hydrogen production. The biological composite system breaks through the limitation of a traditional single microorganism and material hydrogen production system, bismuth stannate (BSO) nanoparticles and escherichia coli are combined through electrostatic interaction, and a novel composite system with the microbial metabolism hydrogen production capacity and the nanometer material photocatalytic performance is constructed. The system creatively integrates the inherent hydrogen production metabolic pathway in escherichia coli cells and the excellent visible light absorption capacity and photocatalytic electron transfer performance of the BSO nano material, and solves the problems that the light energy utilization rate of pure microorganisms is low, and hydrogen production of pure photocatalytic materials depends on exogenous electron donors. Experiments prove that under visible light irradiation, the hydrogen yield of the composite system reaches 0.8 mmol within 4 hours, the hydrogen production efficiency is improved by 1.5 times compared with that of pure escherichia coli, and the efficiency bottleneck of an existing biological hydrogen production technology is remarkably broken through.

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 present invention provides a targeted antibacterial peptide HI-WK targeting Escherichia coli K88, its preparation method and applications, belonging to the field of biotechnology, and its sequence is as shown in SEQ ID No.1. The peptide segment HI with affinity for Escherichia coli was screened from a phage display heptapeptide library. On the basis of this affinity sequence, an active peptide WK was added, and its sequence is as shown in SEQ ID No.3, to improve the penetration of the Escherichia coli cell membrane, resulting in the antibacterial peptide HI-WK. Biological safety detection found that the minimum inhibitory concentration of the antibacterial peptide HI-WK against Escherichia coli K88 was increased by 2 times compared with the active peptide WK, while the geometric mean of the minimum inhibitory concentrations against other Escherichia coli decreased by 1.78 times. In addition, the antibacterial peptide HI-WK did not show hemolytic activity at a concentration of 128 μM and has high application value.

The invention provides a crushing method of escherichia coli genetically engineered bacteria and application thereof, and relates to the technical field of cell disruption, and the crushing method comprises the following steps: dissolving the escherichia coli genetically engineered bacteria in a crushing buffer solution, and crushing in a high-pressure homogenization manner to obtain a crushed bacteria solution. The technical problems that in the prior art, the escherichia coli cell breaking efficiency is low, and a target product is prone to being damaged are solved, and the technical effects that operation is easy and easy to implement, blockage of the high-pressure homogenizer is avoided, the breaking efficiency is remarkably improved, damage to the target product is effectively reduced, and applicability is wide are achieved.

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.

This disclosure provides a method of producing full-length antibodies in E. coli with a yield of at least about 200 mg / L. The E. coli cells typically have an oxidative cytoplasm, which is helpful for maintaining the three-dimensional structure and stability of proteins having disulfide bonds. In some cases, the molar ratio of the produced HC and LC amount from culturing the E. coli ranges from about 1:1 to about 1:3 (e.g., from about 1:1 to about 1:2.5, from about 1:1 to about 1:2).

The invention discloses a method for improving recombinant secretory expression of aprotinin, which effectively promotes expression and secretion of recombinant protein to periplasmic space of escherichia coli by optimizing fusion protein coding gene of pelB signal peptide and antibacterial peptide, and improves the correct folding rate of target protein while improving the expression quantity so as to improve the enzymatic activity of the target protein. The method can be applied to recombinant preparation of aprotinin or aprotinin-containing products.

The invention discloses a method for efficiently synthesizing violacein by using a cell-free system, which comprises the following steps: firstly, constructing recombinant plasmids of key enzymes VioA, VioB, VioC, VioD and VioE in a violacein synthesis route, artificially and exogenously adding substrates, energy, cofactors and the like required by a synthesis reaction based on an escherichia coli cell lysis buffer rich in VioA, VioB, VioC, VioD and VioE, and efficiently synthesizing violacein in vitro. According to the method, the cell-free system is applied to in-vitro synthesis of violacein for the first time, the substrate transfer distance and the distance between multiple enzymes are shortened, and the reaction rate and the catalytic efficiency are improved; operation is simple, stability is high, and reaction conditions are accurate and easy to control; the method has the advantages of simple operation, few side reactions, high production rate and high product yield, can conveniently and quickly detect violacein, and is suitable for detection and verification.