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Double starter cascade regulation gene expression vector and construction method and application thereof

A technology of expression vector and gene expression cassette, which is applied in the field of dual-promoter cascade regulation gene expression vector and its construction, can solve the problem of low ability to induce protein expression, and achieve the effect of improving sensitivity

Inactive Publication Date: 2019-05-21
CHINESE RES ACAD OF ENVIRONMENTAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In existing studies, most of the single promoter systems are used to regulate the expression of reporter genes. The ability of these specific promoter systems to induce protein expression is far lower than that of commonly used powerful promoters such as tac promoter, P L Promoter, T7 promoter, etc.

Method used

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  • Double starter cascade regulation gene expression vector and construction method and application thereof
  • Double starter cascade regulation gene expression vector and construction method and application thereof
  • Double starter cascade regulation gene expression vector and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Construction of dual promoter cascade regulation fluorescent protein gene expression vector As-T7R-sfGFP

[0063] In this example, the T7 RNA polymerase gene was first cloned to the downstream of the arsenic promoter Pars in the vector regulated and expressed by the arsenic promoter Pars, and then the fluorescent protein gene was cloned to the downstream of the T7 promoter of the pET-17b vector, and then the T7 The promoter-fluorescent protein gene sequence was cloned into the arsenic vector, and finally a recombinant vector for strong expression of fluorescent protein induced by arsenic and regulated by a double promoter cascade was formed.

[0064] 1.1 Construction of arsenic promoter-T7 RNA polymerase gene vector (As-T7R-BTT vector)

[0065] 1.1.1 PCR amplification and T / A cloning of T7 RNA polymerase gene fragment

[0066] (1) Extraction of E.coli BL21(DE3) genome: Take 1.5ml of E.coli BL21(DE3) bacterial liquid cultivated overnight, and use bacterial gen...

Embodiment 2

[0112] Example 2 Analysis of Fluorescent Protein Expression Ability of Dual-Promoter Cascade Regulating Fluorescent Protein Gene Expression Vector As-T7R-sfGFP

[0113] 2.1 Dual-promoter cascade regulation fluorescent protein gene expression vector As-T7R-sfGFP transformed strain

[0114] The dual-promoter cascade regulation fluorescent protein gene expression vector As-T7R-sfGFP constructed in the example was transferred into E.coli TOP10 competent cells, and the transformed competent cells were spread on LB containing chloramphenicol On agar medium, leave the plate at room temperature until the liquid is absorbed. Invert the plate and incubate at 37°C, colonies may appear after 12 to 16 hours. The obtained colony clone is the As-T7R-sfGFP transformed strain of the dual-promoter cascade regulation fluorescent protein gene expression vector.

[0115] 2.2 Dual-promoter cascade regulation of fluorescent protein gene expression vector As-T7R-sfGFP fluorescent protein expression...

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Abstract

The invention provides a double starter cascade regulation gene expression vector and a construction method and application thereof. The double starter cascade regulation gene expression vector carries a nucleic acid construct, the nucleic acid construct comprises two gene expression cassettes with cascade regulation relation, the first expression cassette is driven by an inducible starter, and the second expression cassette is driven by a strong starter; wherein the inducible starter is induced and expressed by a specific substance, and a downstream gene coding of the inducible starter can regulate a regulatory element capable of regulating and controlling the expression of the strong starter; and the downstream gene of the strong starter is a target gene. The successful establishment ofthe double starter cascade regulation and control mode not only provides a new method and a new idea for improving the sensitivity of genetic engineering bacteria to environmental pollutants, but alsoenables the high-efficiency amplification effect of the cascade control mode on reporter gene expression to be applied to research of screening analysis of starters or interaction of proteins and thelike, and new technical convenience for gene expression control research is provided.

Description

technical field [0001] The invention belongs to the technical field of environmental biology, and in particular relates to a dual-promoter cascade regulation gene expression vector and its construction method and application. Background technique [0002] Some microorganisms live in an environment containing a certain chemical substance for a long time, and their genome contains specific induction genes, degradation genes or resistance genes for the chemical substance. The reporter gene is fused with these genes, and the induction gene, degradation gene or resistance gene is used as a promoter to regulate the expression of the reporter gene, and then introduced into a selected suitable host bacterium to form a genetically engineered bacterium for environmental monitoring. When the targeted chemical substance acts on the genetically engineered bacteria, it can induce the expression of the reporter gene, and the concentration change or biological toxicity of environmental poll...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/70C12N15/65C12N1/21C12Q1/6897C12R1/19
Inventor 李琴王菲菲陈世武张艳平车飞赵鑫王延红朱静
Owner CHINESE RES ACAD OF ENVIRONMENTAL SCI
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