System for inducing foreign genes to express in gram-negative bacteria and application of system

A Gram-negative bacteria and exogenous gene technology, applied in the biological field, can solve the problems of restricting the production of industrial fermentation products, lack of high-efficiency induced expression gene protein expression system, etc.

Active Publication Date: 2016-07-20
TSINGHUA UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the molecular biology operation of potential industrial strains is still in the initial stage, and the lack of an expression system that can efficiently induce the expression of gene proteins restricts the production of more industrial fermentation products in strains

Method used

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  • System for inducing foreign genes to express in gram-negative bacteria and application of system
  • System for inducing foreign genes to express in gram-negative bacteria and application of system
  • System for inducing foreign genes to express in gram-negative bacteria and application of system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0131] Example 1, Preparation of High-efficiency Inducible Expression System for Exogenous Genes

[0132] 1. Efficient inducible expression system components

[0133] The high-efficiency inducible expression system elements include regulatory elements containing repressor protein expression cassettes and RNA polymerase expression cassettes and exogenous gene expression cassettes;

[0134] The RNA polymerase in the RNA polymerase expression cassette matches the promoter driving the expression of the exogenous gene in the exogenous gene expression cassette;

[0135] RNA polymerase is K1F, VP4 or MmP1;

[0136] The promoter that drives the expression of exogenous genes matched with K1F is K1F-P;

[0137] The promoter that drives the expression of exogenous genes matched with VP4 is VP4-P;

[0138] The promoter that drives the expression of foreign genes matched with MmP1 is MmP1-P;

[0139] The regulatory element containing the repressor protein expression cassette and the RN...

Embodiment 2

[0197] Example 2. High-efficiency inducible expression system induces expression of foreign genes

[0198] The promoters driving the expression of exogenous genes can be constructed on the same or different plasmids in the recombinant bacteria expressing the regulatory elements, or constructed on the genome of the recombinant bacteria themselves that express the regulatory elements of the strain.

[0199] 1. The form of the promoter

[0200] A. The promoter and its driven exogenous gene are expressed in the recombinant strain of the regulatory element in the form of a plasmid

[0201] 1. Construction of recombinant bacteria expressing foreign genes

[0202] The recombinant vector P321-K1FP-GFP was transformed into Halomonas-K1F by conjugative transformation (described above), and the recombinant strain TD1 / K1FP-GFP: LACIK1F was obtained;

[0203] The recombinant vector p321-VP4P-GFP was transformed into Halomonas-VP4 by conjugative transformation (described above), and the r...

Embodiment 3

[0292] Example 3. High-efficiency inducible expression system induces the expression of genes related to cell elongation and shape

[0293] The cell elongation shape-related operon is MinCD, which contains genes MinC and gene MinD in the genome of Halomonas sp. Long (TanD, WuQ, ChenJC, ChenGQ. Engineering Halomonas TD01 for the low-cost production of polyhydroxyalkanoates. MetabEng. 2014Sep16; 26C:34-47.). It is slightly expressed in wild-type Halomonas, so it needs to be overexpressed. This example includes but is not limited to using the MmP1 expression system to overexpress it.

[0294] See SEQ ID NO: 7 for the nucleotide sequence of the MinCD operon, see SEQ ID NO: 11 for the amino acid sequence of the expressed MinC gene, and see SEQ ID NO: 12 for the amino acid sequence of the expressed MinD gene.

[0295] The MinCD operator sequence was used to replace GFP as a foreign gene for high-efficiency induced expression.

[0296] 1. The promoter and the exogenous gene driven...

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Abstract

The invention discloses a system for inducing foreign genes to express in gram-negative bacteria and application of the system. The invention provides a method for expressing the foreign genes in the gram-negative bacteria. According to the method, a regulating and control element comprising a repressor protein expression box and an RNA (ribonucleic acid) polymerase expression box is used for regulating and controlling the expression of a foreign gene expression box in the gram-negative bacteria. Experiments prove that the system for inducing the foreign genes to effectively express in the gram-negative bacteria provided by the invention consists of two parts including specific polymerase and a corresponding promoter; the polymerase, the promoter and driven foreign genes are induced to express in the gram-negative bacteria; the efficient mass expression of the foreign genes can be realized; the important significance of simplifying the extraction step, improving the protein expression quantity and reducing the production cost are realized in industrial production; wide application prospects are realized; wide commercial utilization values are realized.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a system for inducing the expression of foreign genes in Gram-negative bacteria and its application. Background technique [0002] In the fermentation industry, the transformation of industrial microorganisms is one of the basic methods to improve fermentation efficiency, obtain new products, and reduce fermentation costs. The purpose of transformation is mainly to increase the growth rate of microorganisms, increase the expression efficiency of microorganisms, and improve the conversion rate of products. Therefore, the chassis modification of microorganisms and the construction of efficient expression systems in strains have become effective means to achieve this goal. [0003] Among the existing chassis microorganisms, Escherichia coli with T7 expression system and induced by IPTG is one of the most commonly used fermentation microorganisms. Many proteins are induced and expressed...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N1/21C12R1/01C12R1/38
CPCC07K14/195C07K14/245C07K14/28C12N9/1247C12N15/74C12N2800/101C12N2800/60C12N2840/002C12Y207/07006
Inventor 陈国强赵晗张浩千兰陆红
Owner TSINGHUA UNIV
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