9 results about "TetR" patented technology

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 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.

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 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.

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 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.