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A tetracycline-inducible promoter and its preparation method and application

A technology of promoter and tetracycline, applied in the field of tetracycline-induced promoter and its preparation, can solve the problems of inability to apply, lack, and not yet constructed inducible promoter to regulate gene transcription, etc., and achieve the effect of transformation

Active Publication Date: 2021-11-02
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, promoters for heterologous gene expression are limited to constitutive promoters (such as Pgpx), and no inducible promoters have been constructed to regulate gene transcription
In addition, the Halophilic Alkalophilic Sulfur Oxidizer lacks sugar molecule transporters on the cell membrane, so traditional lactose, galactose or xylose inducible promoters cannot be used in the strain

Method used

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  • A tetracycline-inducible promoter and its preparation method and application
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  • A tetracycline-inducible promoter and its preparation method and application

Examples

Experimental program
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Effect test

Embodiment 1 4

[0080] Example 1 Preparation of Tetracycline Inducible Promoter

[0081] (1) Utilize the primers shown in SEQ ID NO.4-5 to amplify the halophilic alkaliphilic sulfalkalivibrio pluripotent D301 genome to obtain the glutamate peroxidase promoter (Pgpx), and introduce restriction enzyme cleavage sites KpnI / BamHI, after electrophoresis gel recovery, digested for 2 hours, and digested the broad host vector pBBR1MCS-1 at the same time, connected the two digested products with T4 ligase overnight at 16°C, and transformed the ligated product pBBR-Pgpx-RFP into the large intestine In Bacillus T1, spread on a chloramphenicol-resistant plate, and perform colony PCR detection after culturing for 16 hours, and obtain positive bacteria;

[0082] (2) if figure 1 As shown, the core promoter region, -35 region and -10 region of Pgpx are predicted in combination with BDGP and BPROM, and the primers shown in SEQ ID NO.6-7 are used to carry out polyoligonucleotide mutations to the spacer sequenc...

Embodiment 2 4

[0084] Example 2 Tetracycline-induced promoter regulates streptomycin resistance gene sm r expression

[0085] (1) Utilize the primers shown in SEQ ID NO.15-16 to amplify the tetracycline-inducible promoter obtained in Example 1, respectively introduce restriction sites KpnI / BamHI at the 5' and 3' ends, digest for 2 hours, and simultaneously Digested expression vector pBBR1MCS-1-sm r , the two digested products were ligated overnight at 16°C with T4 ligase, and the ligated product was transformed into Escherichia coli Sm10. The expression vector of the child (pBBR1MCS-1-Pgpx(tetO)-sm r ) into the halophilic and alkaliphilic sulfoalkali pluripotent D301;

[0086] (2) Culture containing pBBR1MCS-1-Pgpx(tetO)-sm r The halophilic alkaliphilic sulfoalkali Vibrio pluripotent D30116h was streaked on the resistance plates containing aTc+, sm+ and aTc-, sm+, respectively, where aTc is tetracycline and sm is streptomycin.

[0087] As a result, it was found that yellow streaked colo...

Embodiment 3 4

[0088]Example 3 Tetracycline-induced promoter regulates expression of toxin protein mazF

[0089] (1) Utilize the primers shown in SEQ ID NO.15-16 to amplify the tetracycline-inducible promoter obtained in Example 1, respectively introduce restriction sites KpnI / BamHI at the 5' and 3' ends, digest for 2 hours, and simultaneously The expression vector pBBR1MCS-1-mazF was digested, and the two digested products were ligated overnight at 16°C with T4 ligase, and the ligated product was transformed into E. coli S17. B) The expression vector (pBBR1MCS-1-Pgpx(tetO)-mazF) containing the tetracycline-inducible promoter shown was transferred into the halophilic halophilic sulfoalkali pluripotent Vibrio D301;

[0090] (2) Cultivate the halophilic alkaliophilic sulfoalkali Vibrio pluripotent D301 containing pBBR1MCS-1-Pgpx(tetO)-mazF for 16 hours, and streak on the resistance plates containing aTc+, cm+ and aTc-, cm+ respectively, where aTc For tetracycline, cm for chloramphenicol.

[...

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Abstract

The present invention provides a tetracycline-inducible promoter and its preparation method and application. The promoter is to mutate the spacer sequence of a constitutive promoter into a tetracycline operator gene; wherein, the spacer sequence includes the -35 region to the -10 region Any one or a combination of at least two of the nucleic acid sequence between the -10 region and the transcription start site, or the nucleic acid sequence between the -(55±3) bp and the -35 region. In the present invention, the spacer sequence of the constitutive promoter is mutated into a tetracycline operator gene, and the specific combination of the tetracycline operator gene and the tetracycline repressor protein is utilized, so that the constitutive promoter is induced and regulated by tetracycline, and the expression regulation of the target gene by tetracycline is realized. ; The expression vector including the tetracycline-inducible promoter and the target gene is introduced into the halophilic and alkaliphilic sulfoalkali pluripotent, which efficiently promotes the expression of the target gene, and realizes the transformation of the halophilic halophilic sulfoalkali pluripotent sulfoalkalivibrio.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a tetracycline-inducible promoter, its preparation method and application. Background technique [0002] The halophilic alkaliphilic sulfoalkaline pluripotent is a high GC-content Gram-negative and aerobic photosynthetic sulfur bacterium that can + Grow in the environment of 0-4M. It is a chemoautotrophic microorganism that uses carbon dioxide as a carbon source, sulfur substances such as sodium sulfide or sodium thiosulfate as electron donors, and produces elemental sulfur or sulfate in the process of oxidizing sulfur substances. Compared with other biological desulfurization microorganisms, the halophilic and alkaliphilic pluripotent Sulfuralkali Vibrio is tolerant to high-salt and high-alkali environments, which overcomes the defect of low desulfurization rate of traditional desulfurization microorganisms, but has the disadvantage of slow growth rate, increasing oxygen The amount ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/74C12N1/21C12P21/00C12R1/63
CPCC12N15/113C12N15/74C12P21/00
Inventor 邢建民郝学密杨茂华穆廷桢江启沛
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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