57 results about "Operon" patented technology

The invention provides a valine production strain and a construction method and application thereof, a designed acetohydroxy acid synthase mutant is that the 88th basic group of an ilvB gene is changed from a to c, the 382nd basic group is changed from a to g, the 413th basic group is changed from c to t, the gene sequence of a designed artificial operon comprises a promoter, an ilvB (A138V) gene or ilvB (Q30K, S128G, A138V) gene of coding mutated acetohydroxy acid synthase, and an ilvN (G20D, I21D, I21D, I21D, I21D, I21D, I21D, I21D) gene. I22F) gene, a pyk gene for coding pyruvate kinase, and a terminator; by designing a specific acetohydroxyacid synthase mutant and related biological materials and artificial operon, the strain constructed by directional modification of the strain by using a pK18mobsacB system gene editing technology based on allele exchange has the advantages of good genetic stability, high fermentation yield and the like, and valine can be stably produced.

The invention provides a valine production strain as well as a construction method and application thereof, the strain is constructed by utilizing a CRIPSR / Cas9 system gene editing technology based on allele exchange, an artificial operon is designed and constructed, the artificial operon is constructed at a ygaY site, a strong promoter Ptrc is used for controlling ilvIH (G14N S17F)-pykF to carry out synchronous multi-copy expression, and the valine production strain can be used for producing valine. On the basis of the strain, an artificial operon is constructed at an ycgH site, a strong promoter Ptrc is used for controlling ilvC-ilvD to carry out synchronous multi-copy expression, meanwhile, feedback inhibition of valine on ilvIH is relieved, and an ilvIH natural promoter is replaced by the strong promoter Ptrc, so that more pyruvic acid flows to valine; the constructed strain does not contain plasmids, has no defects, does not need induction, has the advantages of good genetic stability, high fermentation yield and the like, and can be used for efficiently synthesizing valine from the beginning by taking glucose as a substrate.

The invention provides an L-lysine production strain as well as a strain construction method and application. A hom (V59A) is introduced on a genome by utilizing a homologous recombination gene editing technology to weaken a competitive approach of lysine; the lysC (T311I) which is not inhibited by L-lysine is overexpressed; the method comprises the following steps of: overexpressing lysE, lysA, ddh, dapB and asd, knocking out a lysI gene and multi-copying the lysE, carrying out synchronous multi-copying on the dapA and EcdapB genes through a manual operon P1676-EcdapB-Pa12-dapA, and carrying out synchronous multi-copying on the dapB, ddh, lysA and lysO genes through a manual operon Psod-dapB-ddh-Ptuf-lysO-lysA; the constructed strain does not contain plasmids, has no defects, does not need induction, has the advantages of good genetic stability, high fermentation yield and the like, and can be used for stably producing L-lysine.

The present invention relates to nucleic acids and modified immune cells or precursors thereof, comprising a first polynucleotide encoding an NF-κB p65-rTetO fusion protein comprising one or more transactivation domains (TAD) of human NF-κB p65 fused to a reverse Tet operator (rTetO) binding domain; a second polynucleotide comprising a Tet operator region (TetOR) for providing inducible expression of one or more transgene(s) operatively linked thereoto; and a third polynucleotide comprising a promoter directing transcription of p65-rTetO from a first strand of DNA, where the p65-rTetO is configured to induce transcription of a transgene of interest operatively linked to the TetOR in the presence of tetracycline or doxycycline (Dox) from an opposite strand of DNA.

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.

The invention relates to the technical field of microorganisms, and particularly discloses a construction method and application of a recombinant microorganism. According to the construction method of the recombinant microorganism, compared with an original strain, the expression quantity of threonine operon leader peptide of the recombinant microorganism is reduced, and the expression quantities of glyceraldehyde-3-phosphate dehydrogenase mutant, aspartate kinase / homoserine dehydrogenase I mutant, homoserine kinase and threonine synthase are increased; the glyceraldehyde-3-phosphate dehydrogenase mutant is characterized in that glycine at the 188th site in a glyceraldehyde-3-phosphate dehydrogenase protein sequence is mutated into serine; the aspartate kinase / homoserine dehydrogenase I mutant is characterized in that glycine at the 433rd site in an aspartate kinase / homoserine dehydrogenase I protein sequence is mutated into arginine; the starting strain is escherichia coli. According to the construction method disclosed by the invention, a novel recombinant microorganism with improved threonine yield can be obtained, and a novel method is provided for fermentation production of threonine.

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.

Disclosed herein are programmable bacteria for tumor-targeted immunotherapeutic delivery. In certain embodiments, the programmable bacteria comprise at least one synchronized lysis circuit contained in a single operon which are capable of being further engineered to cyclically produce anti-cancer therapeutic agents including but not limited to nanobodies against immune checkpoint inhibitors and over-expressed markers in cancers, toxins, tumor antigens, cytokines, and chemokines. In some embodiments, the programmable bacteria comprise at least one synchronized lysis circuit contained in a single operon and at least one plasmid producing a therapeutic agent, i.e., at least one plasmid comprising a nucleic acid sequence which encodes a therapeutic agent. The disclosure also provides methods of curing and treating cancer using the programmable bacteria.

The present disclosure relates to an L-arginine-producing microorganism in which the activity of a regulator of nitrate reductase operon expression is attenuated and the activity of glutamine synthetase is enhanced, and to a method for producing L-arginine using same, wherein the L-arginine-producing microorganism, in which the activity of the regulator of nitrate reductase operon expression is attenuated and the activity of glutamine synthetase is enhanced, has been confirmed to exhibit increased L-arginine production capability compared to a parent strain, and thus can be widely used for L-arginine production.

The application discloses a method for identifying Klebsiella pneumoniae gene circadian rhythm expression by using a bioluminescence reporting system. The application constructs a recombinant bioluminescence Klebsiella pneumoniae driven by a metabolic gene malate:quinone oxidoreductase mqo promoter (Pmqo), and directly observes the change rule of bacterial gene expression under entrainment and free-running conditions of temperature cycle by monitoring the luminescence characteristics. The luxCDABE gene operon comprises genes for coding luciferase and substrate synthesis enzyme, wherein luxA and luxB code for heterodimer luciferase, luxC, luxD and luxE synthesize aldehyde as a substrate for the luciferase luminescence reaction. The luciferase catalyzes the oxidation of aldehyde to generate visible blue-green light with a wavelength of 450-490 nm. The Lux bioluminescence reporting system has the advantages of not needing to add an exogenous substrate and not affecting the normal physiological functions of the host, and can be applied to the identification of Klebsiella pneumoniae gene circadian rhythm expression.

This invention provides an L-lysine-producing strain, its construction method, and its application. The strain utilizes homologous recombination gene editing technology to first overexpress... dapA , aspB Introducing mutations ppc (A482V) Knockout amn Multiple copies are synchronized by manually manipulating Pa12-dapB(T1)-Psod-aspB(T1)-Pa16-Ec_lysA. dapB , aspB Ec. from E. coli W3110 lysA Multiple copies are synchronized via the artificial manipulator Pa12-ddh(RBS)-lysE(T1)-Pa16-gapN(RBS)-amtB. ddh , lysE Streptococcus equi subsp. porphyria gapN , amtB The strain is plasmid-free, defect-free, requires no induction, and has advantages such as good genetic stability and high fermentation yield, enabling stable production of L-lysine.

The invention provides a CRISPR (clustered regularly interspaced short palindromic repeats) transposon gene integration system. The CRISPR transposon gene integration system comprises a vector pQCasTns containing transposase and Cas protein, and a vector pDoner containing a functional sequence, the pQCasTns comprises an operon and a T7 terminator which are connected in sequence; the pDoner comprises a functional sequence recognized by transposase and a crRNA sequence expression module of a targeted genome. The CRISPR transposon system provided by the invention solves a series of problems existing in genome editing of halomonas, and can realize multi-target knockout, high-efficiency deletion of long fragments and high-efficiency integration of exogenous genes.

Disclosed herein are polynucleotides comprising sequences encoding coenzyme M synthase (ComF) linked to a heterologous regulatory element and methods of using the same. The polynucleotides may comprise synthetic operons comprising additional sequences encoding enzymes, e.g., a taurine-pyruvate aminotransferase, a sulfoacetaldehyde acetyl transferase, or a sulfopyruvate decarboxylase. Also disclosed herein are recombinant prokaryotic cells, e.g., recombinant bacterial, e.g., E. coli, or archaeal cells, e.g., Methanosarcina acetivorans with improved tolerance to oxidative stress.

The invention provides a salmonella paratyphi A strain for recombining and expressing Vi polysaccharide, which is characterized in that a viaB operon for expressing the Vi polysaccharide from salmonella paratyphi is cloned into a salmonella paratyphi A genome by a genetic engineering technology, so that the expression of the Vi polysaccharide in the salmonella paratyphi A is realized; furthermore, a constitutive strong promoter (Tac promoter) is used for replacing a viaB natural promoter to realize high expression of the Vi polysaccharide under an in-vitro culture condition; further, the msbB gene of the strain is knocked out to reduce the acylation level of the LPS and reduce the toxicity of the LPS; the vesicle vaccine prepared from the strain can induce immune protection effects on two serotypes.

The application discloses an Aeromonas phage recombinant system, an expression vector, a recombinant strain and application thereof, and the recombinant system comprises a recombinase operon 109TES derived from a salmonella Aeromonas phage vB_AsaM-56; the operon comprises at least one gene with exonuclease function and one single-stranded DNA annealing protein gene. Through function verification in Escherichia coli, 109TES can efficiently mediate linear circle recombination and linear linear recombination by using a short homologous arm (50 bp), and has stable and reliable recombination function in a heterologous host, indicating that the recombinase operon from a non-Escherichia coli phage can also exert efficient homologous recombination function in Escherichia coli, and can be used as a powerful candidate for constructing a new type of recombination tool, and provides a new choice for developing a multi-host available gene editing system.

The invention belongs to the field of biology, and discloses a proteus mirabilis homologous recombination system expression plasmid which comprises a pBR322 replication starting point, a resistance gene, an arabinose inducible promoter and a proteus mirabilis-derived homologous recombination operon. The homologous recombination operon is used for coding a TPM66325 protein, an EPM66325 protein, a single-chain binding protein SSB and a fake release protein. The expression plasmid has relatively high recombination efficiency in proteus mirabilis GDMCC 66325 and can also play a recombination role in a proteus mirabilis standard strain ATCC35659, and the expression plasmid is obtained by knocking out a related gene bcsB formed by a hemolysin related gene hpmA and a biological membrane of the proteus mirabilis GDMCC 66325 and directly knocking in a heterologous gene after a related gene cluster is adjusted by flagellum. The effectiveness of the expression plasmid in recombinant operation of proteus mirabilis is verified; meanwhile, the invention further discloses a proteus mirabilis homologous recombination system, application and a gene knockout / knockin method.

The invention provides a DNA marker plasmid as well as a combination system and application thereof, and relates to the field of molecular biology. The DNA marker plasmid is formed by connecting a linearized skeleton sequence and an insertion fragment sequence; the linearized skeleton sequence is derived from a reference plasmid from which a lactose operon sequence is deleted, and a selectable marker gene of the linearized skeleton sequence has a preset recognition site of restriction enzyme; the insertion fragment sequence is formed by connecting a plurality of DNA fragments in series, and recognition sites of restriction endonuclease are arranged between the adjacent DNA fragments. According to the plasmid, a restriction enzyme cutting site is introduced into a selective marker, so that a skeleton is directly used as a characteristic band, and the complete utilization of a sequence is realized. Waste and purification are avoided after enzyme digestion, the cost is remarkably reduced, and the preparation efficiency is improved.

The invention provides a strain for producing L-isoleucine and a construction method and application of the strain. Pck genes of the strain are knocked out, ilvBN (G20D, I21D and I22F), ilvA (V140M, F383A), lysC (T311I), hom (G378I), thrB (A20G), ppc, aspB, thrC, asd and brnFE are over-expressed, and the strain is prepared through operons Psod-ilvBN (G20D, I21D and I22F)-ilvA (V140M, F383A)-Ptuf-thrA-thrB-thrC and Psod-ppc-aspB-Ptuf

Described herein are compositions and methods for generating a chassis for high-capacity production of different classes of high-value terpenoids. The chassis is a microorganism host that expresses a first operon comprising a modified ribosomal binding site, a Hmg-CoA reductase (HmgR) polypeptide, a 3-hydroxy-3-methylglutaryl CoA synthase (HmgS) polypeptide, and a β-ketothiolase (PhaA) polypeptide; and a second operon comprising a modified ribosomal binding site, a mevalonate kinase (MvK1) polypeptide, a phospho-mevalonate kinase (MvK2) polypeptide, and a diphosphomevalonate decarboxylase (MvD) polypeptide.

The invention discloses an HlyA secretion system for increasing the secretion amount of substrate protein and a construction method and application thereof.The HlyA secretion system comprises a first component, a second component and a third component, the first component comprises an inner membrane transporter HlyB and a membrane interstitial adaptor protein HlyD, and the inner membrane transporter HlyB and the membrane interstitial adaptor protein HlyD are obtained by expressing modified hlyBD operons; according to the modified hlyBD operon, a nucleotide sequence for coding a hexa-polyhistidine tag is inserted behind an hlyD gene initiation codon in the hlyBD operon; and a second component: an outer membrane channel protein TolC derived from a host cell. According to the invention, the HlyD protein is specifically modified, so that the secretion amount of the HlyA secretion system to the substrate protein is obviously increased.

The present invention relates to a bio-attenuated Gram-negative bacterium comprising a modified hlyCABD operon, wherein the modified hlyCABD operon is divided into a first segment and a second segment, each segment operably linked to an independently controlled promoter, the first segment comprising a heterologous polynucleotide encoding a cargo molecule upstream of an hlyAs translocation sequence, the heterologous polynucleotide encoding the cargo molecule replacing the hlyA gene, and the second segment comprising an hly gene involved in secretion. Thus, the present invention relates to a bacterial delivery system and various uses and methods thereof.

Disclosed herein are methods and systems to generate novel nucleic acid sequences that bind to an allosteric transcription factor and novel nucleic acid sequences generated by said methods.

A non-resistance plasmid production system constructs a plasmid containing a nucleic acid fragment 3 for coding small RNA, a host cell expression cassette containing a toxic gene expression cassette and an anti-toxic gene expression cassette through combined design of a toxic transcript / anti-toxic transcript, an operon related element and the small RNA. The toxic gene expression cassette comprises a promoter and a nucleic acid fragment 1 for transcribing a toxic transcript, and the anti-toxic gene expression cassette comprises a promoter, an operon and a nucleic acid fragment 2 for transcribing an anti-toxic transcript. Through reverse complementation of the nucleic acid fragment 3 and the nucleic acid fragment 1, a steric effect is generated, and a toxic transcript is inhibited, so that the screening production of plasmids can be realized without adding antibiotics or other inhibitors in the whole plasmid production process, and the harm of the antibiotics and the inhibitors to the plasmids in cell gene therapy is solved.

The present invention relates to the production of a recombinant plasmid as a vector for introducing repair sequences into nitrogen-fixing bacterial cells incapable of expressing nitrous oxide reductase. Successful insertion of the recombinant plasmid leads to restoration of the inactive operon, preventing the release of the greenhouse gas nitrous oxide (N₂O) during the denitrification process. The resulting mutant strain exhibits the same agronomic efficiency as the original strain, but with the added benefit of significantly reducing N₂O emissions, contributing to a more sustainable approach to agriculture and food production. Additional aspects of the present invention relate to primer pairs, nucleotide sequences, modified microorganisms, as well as methods for the preparation thereof.

The present invention relates to a genetically modified Clostridium acetobutylicum able to grow on lignocellulosic biomass, in which the cip-cel operon is overexpressed, the expression level of each gene of the cip-cel operon in the genetically modified Clostridium acetobutylicum being higher than its expression level in a corresponding non-genetically modified Clostridium acetobutylicum, as well as derivatives thereof with further modifications increasing growth on lignocellulosic biomass, and the use of these strains for the production of bulk chemicals by conversion of lignocellulose as source of carbon, such as ethanol, butanol, glycerol, 1,2-propanediol, acetone, isopropanol, isobutene, hydrogen, acetic acid and lactic acid and the like.

The invention belongs to the technical field of gene engineering, and relates to a recombinant plasmid, a recombinant system and a construction method and application thereof. The plasmid expresses an operon under the induction of a promoter, and the operon is composed of a CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-associated 9) system and a Red / ET recombinase system derived from pseudoalteromonas P. agarivorans Hao 2018. The recombinase system comprises a recombinase 2018Red [beta] and a recombinase 2018Red [alpha]; homologous arms with different lengths for recombination are inserted between the Cas9 protein and the recombinase 2018Red alpha beta. According to the invention, the homologous arm and the guide RNA sequence are constructed to the same site of the plasmid, and the construction can be completed by only one step of Gibson assembly, so that the workload is reduced, the working efficiency is improved, and the gene editing period can be completed in a relatively short time. The invention provides a new tool for gene editing, and has a wide application prospect.

The present invention relates to the production of a recombinant plasmid as a vector for introducing repair sequences into nitrogen-fixing bacterial cells incapable of expressing nitrous oxide reductase. Successful insertion of the recombinant plasmid leads to restoration of the inactive operon, preventing the release of the greenhouse gas nitrous oxide (N₂O) during the denitrification process. The resulting mutant strain exhibits the same agronomic efficiency as the original strain, but with the added benefit of significantly reducing N₂O emissions, contributing to a more sustainable approach to agriculture and food production. Additional aspects of the present invention relate to primer pairs, nucleotide sequences, modified microorganisms, as well as methods for the preparation thereof.

A composition comprising a recombinant probiotic organism engineered to constitutively express an enzyme involved in maintaining telomere length, wherein the enzyme comprises a telomerase subunit, the genetic modification comprises an operon comprising a gene sequence encoding telomerase having a promoter recognized by the RNA polymerase of the probiotic organism and a genetic modification that induces the secretion of telomerase into the host organism, and the genetic modification of the telomerase gene sequence enhances the entry of the telomerase enzyme into the host organism's cells.

The invention discloses an aeromonas bacteriophage recombination system, an expression vector, a recombination strain and application of the aeromonas bacteriophage recombination system. The recombination system comprises a recombinase operon 109TES derived from aeromonas salmonicida bacteriophage vBAsaM-56; the operon comprises at least one gene with exonuclease function and a single-stranded DNA annealing protein gene. Through functional verification in Escherichia coli, 109TES can utilize a short homologous arm (50bp) to efficiently mediate wire loop recombination and wire-line recombination, and has a stable and dependable recombination function in a heterologous host, which indicates that a recombinase operon from a non-Escherichia coli bacteriophage can also exert an efficient homologous recombination function in the Escherichia coli, so that the recombinant enzyme operon can be used for preparing a recombinant vector. The method can be used as a powerful candidate for constructing a novel recombination tool, and a new choice is provided for developing a multi-host available gene editing system.

The invention discloses an epitope peptide of avian leukosis virus J subgroup envelope protein GP85, a hybridoma cell strain 3G10E12C4, a monoclonal antibody (mAb) generated by the hybridoma cell strain 3G10E12C4 and application of the monoclonal antibody (mAb). An ALV-J subgroup GP85 protein FE10 epitope peptide is expressed on the surface of an engineering bacterium DH5alpha through a MisL protein passenger-carrying structural domain of a salmonella V-type secretion system in an exhibiting manner and serves as an immunogen to immunize a BALB / c mouse, meanwhile, the FE10 epitope peptide is inserted into a peg pilus operon, an inert carrier bacterium S9H is introduced, the FE10 epitope peptide serves as a detection system, mouse splenocytes and SP2 / 0 cells are fused, and a detection result is obtained. And the monoclonal antibody is obtained through double screening of ELISA (enzyme-linked immuno sorbent assay) and detection targets. The mAb prepared from the hybridoma cell can be specifically combined with the ALV-J strain infected with the HD-11 cell, and has potential application value and prospect in detection, treatment and purification of the ALV.