Tetracycline regulatory protein mutant gene and application thereof in regulatory gene expression and environmental detection

A technology for regulating proteins and tetracyclines, applied in application, genetic engineering, plant genetic improvement and other directions, can solve the problems of insufficient sensitivity, low broad-spectrum, low detection limit of bacterial biosensors, etc., to improve sensitivity, reduce Dox induction concentration, Achieve the effect of in situ detection

Pending Publication Date: 2022-03-04
ANHUI UNIVERSITY +1
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  • Abstract
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Problems solved by technology

[0010] The purpose of the present invention is to provide a tetracycline regulatory protein mutant gene that can specifically respond to tetracycline antibiotics and its application in environmental detection and regulation of gene expression, so as to solve the problem of bacterial biology based on tetracycline regulatory system construction in the prior art. The detection limit of the sensor is low, the sensitivity is not enough, and the broad spectrum is low; and the technical problem that the tetracycline treatment concentration is too high in the regulation of gene expression in the use of the Tet-off gene expression regulation system has a negative impact on biological experiments

Method used

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  • Tetracycline regulatory protein mutant gene and application thereof in regulatory gene expression and environmental detection
  • Tetracycline regulatory protein mutant gene and application thereof in regulatory gene expression and environmental detection
  • Tetracycline regulatory protein mutant gene and application thereof in regulatory gene expression and environmental detection

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Experimental program
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Effect test

Embodiment 1 4

[0040] Embodiment 1 The acquisition of tetracycline regulation system gene:

[0041] 1), the acquisition of the wild-type tetracycline regulation system gene:

[0042] Using the plasmid pUC57-TetR-sfGFP as a template, PCR amplification was carried out with the primers described in SEQ ID NO: 2 and SEQ ID NO: 3 to obtain the regulatory protein gene TetR, TetR regulatory protein binding site TetO, and an inducible promoter P TetR and a wild-type tetracycline-inducible system for the reporter gene sfGFP,

[0043] 所述质粒pUC57-TetR-sfGFP是根据质粒pFS_0271_pET30_pCat-tetR-Term-ptetA-sfGFP-rrnBterm_Fusicatenibacter_saccharivorans_ARRAY2_FaqI的TetR调控系统部分合成而来的,质粒pFS_0271_pET30_pCat-tetR-Term-ptetA-sfGFP-rrnBterm_Fusicatenibacter_saccharivorans_ARRAY2_FaqI的获得方法参见Schmidt F 2018 paper by et al. (Schmidt, Florian, Mariia Y. Cherepkova Randall J. Platt*. Transcriptional recording by CRISPR spacer acquisition from RNA. Nature 2018; doi:10.1038 / s41586-018-0569-1.).

[0044] Table 1: Primer sequenc...

Embodiment 2

[0060] Dosage Effect Experiment of Evolved Bacterial Biosensors on Dox

[0061] 1) Inoculate the evolved bacterial biosensor on the LB solid medium plate containing kanamycin resistance.

[0062]2) Cultivate overnight at 37°C; at the same time, transform the wild-type tetracycline-inducible recombinant vector pSB1k3-TetR-sfGFP into E.coli DH5α competent cells to obtain wild-type bacterial biosensors, and cultivate wild-type bacterial biosensors simultaneously.

[0063] 3) Pick a single clone, inoculate it in 3 mL of LB liquid medium containing kanamycin resistance, and culture it overnight at 37° C. at 200 rpm to obtain an overnight bacterial liquid.

[0064] 4) Inoculate the overnight bacterial solution from step 2) at 1:20 in fresh LB liquid medium containing kanamycin resistance and expand to logarithmic growth phase (OD 600 =0.4-0.6), to obtain the logarithmic phase bacterial liquid.

[0065] 5) Use deionized water to prepare two sets of tetracycline antibiotic standard ...

Embodiment 3

[0070] Sensitivity experiments of evolved bacterial biosensors

[0071] 1) The evolved bacterial biosensors were inoculated on LB solid medium plates containing kanamycin resistance, cultured overnight at 37°C, and the wild-type bacterial biosensors were simultaneously cultivated.

[0072] 2) Pick a single colony, inoculate it in 3 mL of LB liquid medium containing kanamycin resistance, and culture it overnight at 37° C. and 200 rpm to obtain an overnight bacterial liquid.

[0073] 3) Expand the overnight bacterial solution of step 2) at 1:20 in fresh LB liquid medium containing kanamycin resistance, culture at 37°C, 200rpm shaker to OD 600 =0.4-0.6, to obtain the logarithmic phase bacterial liquid.

[0074] 4) Prepare 200 mg / L Dox stock solution with ultrapure water.

[0075] 5) Add the Dox stock solution to the logarithmic phase bacterial solution obtained in 3), so that the final concentration of the inducer is 200 μg / L, as the induction group; simultaneously take the log...

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Abstract

The invention discloses a tetracycline regulatory protein mutant gene and application thereof in regulation of gene expression and environmental detection, and relates to the field of genetic engineering whole-cell biosensors and gene expression regulation, and the tetracycline antibiotic inducible operon gene has a nucleotide sequence as shown in SEQ ID NO: 1. The tetracycline antibiotic inducible operon is a mutant of wild type tetracycline regulatory protein TetR, can be directly used for optimized construction of a tetracycline antibiotic inducible biosensor, is used for detecting the content of tetracycline antibiotics, is high in detection limit and good in specificity, and can be used for detecting the content of the tetracycline antibiotics. And eight tetracycline antibiotics including tetracycline can be detected. The tetracycline antibiotic inducible operon gene can also be used for gene expression regulation and control, the response performance of the tetracycline antibiotic inducible operon gene to Dox is improved, downstream gene expression can be silenced through low-concentration Dox, the influence of drug treatment on experimental results is reduced as much as possible, and the accuracy of the experimental results is ensured.

Description

technical field [0001] The invention relates to the field of genetically engineered whole bacteria biosensor and gene expression regulation, in particular to a tetracycline regulation protein mutant gene and its application in regulating gene expression and environment detection. Background technique [0002] The tetracycline transcriptional regulator TetR (tetracycline repressor) regulatory protein is derived from the Escherichia coli Tn10 transposon, which controls the expression of the tetracycline overflow pump gene and is related to bacterial drug resistance. TetR can specifically combine with Tet operator (Tetoperator, TetO). The Tet expression regulation system achieves the purpose of regulating the expression of the target protein by inducing drugs (such as tetracycline) to change the conformation of the regulatory protein. When there is no Tet in the cell, TetR will bind to TetO, thereby blocking the expression of downstream resistance genes; when Tet exists, Tet w...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C12N15/70C12N15/65C12Q1/6897C12Q1/02C12R1/19
CPCC07K14/245C12N15/70C12N15/65C12Q1/6897C12Q1/025G01N2333/245
Inventor 吴李君李顺兰陈少鹏陈东东陶诗频徐升敏肖翔
Owner ANHUI UNIVERSITY
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