20 results about "TetR" patented technology

The invention relates to the technical field of biology, in particular to a molecular switch system with a signal cascade amplification function and application of the molecular switch system. The invention provides a molecular switch of a signal cascade method and a gene expression or regulation system. The molecular switch comprises an activator protein araC gene, a bidirectional pTet promoter, a repressor protein tetR gene, a pBad promoter and a tetO operon which are connected from upstream to downstream. When the molecular switch is used for expression or silencing of a target gene, lower background leakage, higher response rate, larger expression range and higher expression intensity are achieved, and expression is more homogeneous; and the inhibition efficiency is obviously improved.

The invention belongs to the technical field of genetic engineering, and particularly relates to a method for increasing the yield of lincomycin by knocking out a streptomyces lincomycin pAgene0296 gene. The method comprises the following steps: knocking out a coding gene pAgene0296 of TetR / AcrR family repressor protein in streptomyces lincomycin, and fermenting the obtained mutant strain to produce lincomycin. Experimental results show that after the pAgene0296 gene of the streptomyces lincomycin is knocked out, the yield of the lincomycin is remarkably increased. The invention further provides a construction method of the pAgene0296 gene deletion type streptomyces lincoli mutant strain, according to the method, the coding gene of the streptomyces lincoli is directionally modified through a gene engineering technology, so that the pAgene0296 gene is knocked out, and the obtained pAgene0296 gene deletion type streptomyces lincoli mutant strain can be used for efficiently producing lincomycin or a product in the synthesis process of the lincomycin.

The invention relates to bacillus subtilis with high yield of beta-carotene as well as a construction method and application of the bacillus subtilis, and belongs to the technical field of fermentation engineering. According to the invention, extracellular proteases AprE, NprE, Bpr, Mpr, Epr and NprB are knocked out from bacillus subtilis, a repressor protein mutant tetR expression cassette and a beta-carotene synthetic pathway related gene expression cassette are constructed, and a tetracycline inducible promoter is used for expressing beta-carotene synthetic pathway related genes crtE, crtI, crtB and crtY, so that the bacillus subtilis for producing beta-carotene is successfully constructed. The recombinant strain G600-crtEBIY is constructed, the induction time and the inducer concentration are optimized in the fermentation process of the constructed recombinant strain G600-crtEBIY, the yield of the optimized beta-carotene in a shake flask fermentation system reaches up to 18.5 mg / L, stable and efficient production of the beta-carotene is achieved, and industrial production and popularization of the beta-carotene are facilitated.

This invention discloses a high-efficiency protein expression system, its construction method, and its applications. The system includes a heterozygous promoter activator and a heterozygous promoter. The heterozygous promoter activator consists of the DNA-binding protein TetR, a linker peptide, and the transcription activator Mit1-Δ60-96. The heterozygous promoter consists of the protein-binding sequence TetO, a spacer sequence, and a basal promoter. The linker peptide is (GGGGS)×2, the spacer sequence is TCGCGCGC, and the basal promoter is P. AOX1 The core promoter of this invention. In the absence of a heterozygous promoter activator, the target protein gene initiated by the heterozygous promoter is expressed very weakly; in the presence of a heterozygous promoter activator, the target protein gene is expressed efficiently. This invention's system has good applicability and scalability, and the expression intensity of various target proteins, including xylanase, phytase, and glucosidase, is significantly higher than that of commonly used P. AOX1 Promoter.

The invention discloses a dynamic regulation and control system, a pantoic acid production strain and a preparation method and application of the pantoic acid production strain. The dynamic regulation and control system comprises a LuxI module, a LuxR module, a tetR module and a tetracycline regulation and control module, wherein the LuxI module comprises a LuxI gene and a promoter M1-93, the LuxR module comprises a LuxR gene and a promoter lpp, the tetR module comprises a tetR gene and a promoter pLux, and the tetracycline regulation and control module comprises a promoter Pteto for down-regulating gltA expression. Experiments prove that the dynamic regulation and control system has an obvious positive effect in the aspect of controlling the biomass accumulation of the pantoic acid producing microorganisms, not only is the problem of overhigh biomass accumulation of pantoic acid engineering bacteria in industrial production solved, but also the yield of pantoic acid is increased, and a foundation is laid for realizing industrial microbial fermentation production of pantoic acid. The method has an important application value.

The invention provides a method for improving the yield of lincomycin by modifying a streptomyces lincomycin SLCG3615 gene, which comprises the following steps: knocking out a TetR family transcriptional regulation gene SLCG3615 in the streptomyces lincomycin through a genetic engineering way to obtain a lincomycin high-yield engineering strain, and fermenting the obtained strain to produce the lincomycin. Wherein the nucleotide sequence of the SLCG3615 gene is as shown in SEQ ID NO. 1. The invention also provides an application of the SLCG3615 gene knockout modified streptomyces lincomus. The lincomycin biosynthesis negative regulator SLCG3615 is screened in the research, the SLCG3615 gene copy on the chromosome of the streptomyces lincomycin is knocked out through a genetic engineering way, a lincomycin high-yield strain can be obtained, and a technical support is provided for improving the fermentation yield of the lincomycin in industrial production.

The invention discloses a construction method and application of a tetracycline induction condition termination replica plasmid. The plasmid comprises the following components: a repB replica gene, a TetR tetracycline transcriptional regulatory factor, a Pet promoter and a replicon gene element. According to the construction method, a chloramphenicol resistance gene cat, a replicating gene pUCmini and a gene segment PNDKR are seamlessly cloned and assembled to obtain plasmids, the method is suitable for multi-round, rapid and multi-site biological genetic engineering, and the next round of operation process is rapidly entered without multi-round culture and screening of plasmid loss strains; the method can be applied to rapid plasmid loss in the lactococcus lactis, and has important significance on future industrial operation of the lactococcus lactis.

The present disclosure relates to cell lines comprising a polynucleotide comprising at least one tetracycline operon repressor protein (TetR) expression cassette for use in the production of recombinant adenovirus (Ad) vectors encoding other toxic transgenes.

This invention discloses a high-efficiency protein expression system, its construction method, and its applications. The system includes a heterozygous promoter activator and a heterozygous promoter. The heterozygous promoter activator consists of the DNA-binding protein TetR, a linker peptide, and the transcription activator Mit1-Δ60-96. The heterozygous promoter consists of the protein-binding sequence TetO, a spacer sequence, and a basal promoter. The linker peptide is (GGGGS)×2, the spacer sequence is TCGCGCGC, and the basal promoter is P. AOX1 The core promoter of this invention. In the absence of a heterozygous promoter activator, the target protein gene initiated by the heterozygous promoter is expressed very weakly; in the presence of a heterozygous promoter activator, the target protein gene is expressed efficiently. This invention's system has good applicability and scalability, and the expression intensity of various target proteins, including xylanase, phytase, and glucosidase, is significantly higher than that of commonly used P. AOX1 Promoter.

The present invention belongs the technical field of biomacromolecule detection. Provided are a cell indicator system, and a preparation method therefor and the use thereof. The cell of the cell indicator system comprises at least one of a vector A, a vector B and a vector C; the vector A comprises a gene encoding an TetR protein; the vector B comprises genes encoding a biotin acceptor peptide and ubiquitin; and the vector C comprises genes encoding a targeting E3 ligase and a biotin ligase. By means of constructing the cell line capable of expressing the TetR protein, biotin acceptor peptide, ubiquitin, targeting E3 ligase and biotin ligase, and inducing the expression by means of doxycycline, the targeting E3 ligase can bind to a substrate, thereby ubiquitinating and biotinylating the substrate, and thus a known ubiquitinated substrate can be rapidly detected and an unknown ubiquitinated substrate can be rapidly identified.

The invention relates to a cell indicator system as well as a preparation method and application thereof, and belongs to the technical field of biomacromolecule detection. According to the cell indicator system, cells comprise at least one of a carrier A, a carrier B and a carrier C; the carrier A contains a TetR protein coding gene; the carrier B contains a biologically acceptable peptide and a coding gene of ubiquitin; and the vector C contains coding genes of targeted E3 ligase and biotin ligase. According to the application, the cell line capable of expressing the TetR protein, the biological accepting peptide, the ubiquitin, the targeted E3 ligase and the biotin ligase is constructed, the targeted E3 ligase can be combined with the substrate through the induced expression of the doxycycline, so that the substrate is ubiquitinated and biotinylated, and the known and unknown ubiquitinated substrates can be rapidly detected and identified.

The invention relates to a transcription factor-based cell-free biosensor and an application thereof in water environment antibiotic detection. Firstly, a cell-free biosensor containing TetR or MphR is constructed, the cell-free biosensor comprises a DNA template, a transcription factor TetR or MphR, RNA polymerase and a DFHBI-1T fluorescent probe, the DNA template comprises a reporter gene capable of being combined with the fluorescent probe, and an operon corresponding to the transcription factor TetR or MphR is arranged on the upstream of the reporter gene; the biosensor can respond to tetracycline or macrolide antibiotics, and further, the content of the antibiotics can be measured according to the change of fluorescence intensity in a cell-free biosensor system. The invention provides the cell-free biosensor with high sensitivity and high stability, and the biosensor can realize high-efficiency detection under different water body conditions and realize stronger environmental adaptability.

The present invention discloses a method for improving the fermentation yield of a heterologous strain of spinosad by modifying a Streptomyces albus B4 chassis. The method comprises the following steps: overexpressing a TetR family transcriptional regulatory factor XNR_0706 involved in regulating fatty acid β-oxidation from S. albus B4 through a genetic engineering approach, obtaining a chassis strain with increased polyketide compound precursor acyl-CoA, and heterologously expressing a spinosad gene cluster in the modified chassis strain to achieve increased fermentation yield of spinosad. Furthermore, when exogenous fatty acids are added, the yield of spinosad is further increased, thereby providing technical support for increasing the yield of spinosad in industrial production.

This invention provides a method for modifying Streptomyces lincosinate. SLCG_3615 A method to increase lincomycin production through gene therapy involves knocking out the TetR family transcriptional regulatory genes in Streptomyces lincomyces via genetic engineering. SLCG_3615 A high-yield engineered strain of lincomycin was obtained, and the obtained strain was used to produce lincomycin through fermentation; among which, SLCG_3615 The nucleotide sequence of the gene is shown in SEQ ID NO.1. This invention also provides... SLCG_3615 Applications of gene knockout modified *Streptomyces lincomycin*. In this study, the negative regulator of lincomycin biosynthesis, SLCG_3615, was screened and knocked out on the chromosome of *Streptomyces lincomycin* via genetic engineering. SLCG_3615 SLCG_3615 Gene copying can yield high-yield strains of lincomycin, providing technical support for increasing the fermentation yield of lincomycin in industrial production.

The invention provides a parent cell comprising at least one nucleoprotein complex comprising at least one DNA-binding scaffold tethered to at least one control plasmid; and wherein the at least one n

The present invention provides a photoactivatable Tet-OFF / ON system that can precisely control temporal and spatial gene expression. The present invention is a PA-Tet-OFF / ON system that includes a target gene expression cassette including a TRE having a TetO sequence, a promoter which is controlled by the TRE, and a target gene whose expression is controlled by the promoter; a first fusion protein expression cassette containing a gene which encodes a first fusion protein containing TetR or rTetR and a first protein; and a second fusion protein expression cassette containing a gene which encodes a second fusion protein containing p65AD and a second protein, in which the first protein and the second protein bind to each other to form a heterodimer only in a state of being irradiated with light at a specific wavelength.

The invention discloses a preparation method of a tGmR bacterial sensor, a product of the tGmR bacterial sensor and application of the tGmR bacterial sensor. The tGmR bacterial sensor contains recombinant plasmids pET-mpR-tetR-mpA-tetM. The biosensor disclosed by the invention can realize real-time, low-cost and biological related detection on chemical pollutants; the genetic stability is better; resistance genes tetM and mpphA are introduced, so that the detection sensitivity and the tolerance to high-concentration antibiotics are improved; on the basis of bacteria, the bio-available concentration of tetracycline and macrolide antibiotics can be detected at the same time; the detection limit of the tGmR bacterial sensor on tetracycline is 11.1 [mu] g / L, the LOD of the tGmR bacterial sensor on erythromycin is 16.3 [mu] g / L, and in addition, the tGmR bacterial sensor can simultaneously detect the erythromycin with the maximum of 125 [mu] g / L and the tetracycline with the maximum of 250 [mu]

The invention belongs to the technical field of synthetic biology and genetic engineering, and particularly discloses an anti-leakage Cre recombinase expression cassette and application thereof in pichia pastoris gene editing. The expression cassette is composed of lox71-selection marker-sh blele-Cre coding sequence (SEQ ID NO: 1)-TetO manipulation sequence-lox66, wherein the Cre coding sequence (SEQ ID NO: 1) is optimized by pichia pastoris codon and is introduced into escherichia coli rare codon, and IIS type restriction enzyme sites such as PaqCI, BsmBI, BsaI, AarI and the like are completely eliminated; the gene is cloned to a pPICZ skeleton and introduced into an escherichia coli host of chromosome integrated tetR, Cre leakage expression can be thoroughly inhibited in a large-scale amplification stage, and recombination toxicity and plasmid mutation are avoided. An'upstream homologous arm-expression cassette-downstream homologous arm 'edit box can be obtained through one-step assembly by using Golden Gate, and construction is completed within 3 days; electrically transforming pichia pastoris after linearization, and integrating efficiency gt for the first time; according to the method disclosed by the invention, the content of the resistance marker is 70%, the resistance marker removal rate is 100% through methanol-induced Cre self-deletion, and only one lox71 / 66 heterozygous site is reserved. According to the invention, the problems that the traditional Cre / loxP system is easy to mutate in escherichia coli and low in efficiency in yeast are solved, and an efficient and safe universal gene editing tool is provided for industrial microbial metabolism engineering.

This invention relates to the field of genetic engineering technology and provides a method for increasing lincomycin production by modifying the SLCG_1979 gene in *Streptomyces lincomyces*. The method involves deleting the SLCG_1979 gene, which encodes a TetR family transcription factor, from the *Streptomyces lincomyces* strain through genetic engineering, thereby obtaining a high-yielding lincomycin-producing strain. The resulting strain is then used for fermentation to produce lincomycin. The nucleotide sequence of the SLCG_1979 gene is shown in SEQ ID NO. 1. This invention also provides an application of the above-mentioned method for increasing lincomycin production in industrial strains. The advantage of this invention is that deleting the SLCG_1979 gene on the *Streptomyces lincomyces* chromosome through genetic engineering can yield a high-yielding lincomycin-producing strain, providing technical support for increasing lincomycin fermentation yield.

The invention discloses a method for constructing an escherichia coli whole genome CRISPRi library and an application of the escherichia coli whole genome CRISPRi library in target product synthesis, and belongs to the technical field of gene engineering. According to the invention, an sgRNA plasmid library is designed according to escherichia coli BL21 (DE3) genome information, and a sensor plasmid and an sgRNA library plasmid are simultaneously introduced into a tetR-Ptet-dCas9 expression strain to obtain a CRISPRi library which can be used for screening strains with high yield of target products. According to the present invention, by using lactyl-N-neotetraose as a model product, eight inhibition genes, such as cysW, pyrG, sprE, pcnB, YqjA, TnaB, TolB and ihfB, beneficial to the production of the lactyl-N-neotetraose are obtained; according to the invention, a screening method with feasibility is established, a key gene beneficial to synthesis of a target product is identified and obtained, and an efficient method is provided for enhancing synthesis of the target product of a strain.