Escherichia coli membrane protein ZraS mutant, gene for coding mutant, recombinant vector as well as preparation method and application of Escherichia coli membrane protein ZraS mutant

A technology of Escherichia coli and mutants, applied in the field of genetic engineering, can solve the problems of complex components of samples to be tested, difficulty in achieving detection targets in wild-type systems, inability to specifically distinguish lead ions and zinc ions, etc., achieving low cost and improved response sensitivity , the effect of simple operation

Pending Publication Date: 2022-03-04
GUANGZHOU INST OF ADVANCED TECH CHINESE ACAD OF SCI
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Problems solved by technology

However, in the actual application process, the composition of the sample to be tested is often complex, and two ions may exist at the same time, and the system cannot specifically distinguish lead ions and zinc ions
On the other hand, according to literature reports, the application of

Method used

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  • Escherichia coli membrane protein ZraS mutant, gene for coding mutant, recombinant vector as well as preparation method and application of Escherichia coli membrane protein ZraS mutant
  • Escherichia coli membrane protein ZraS mutant, gene for coding mutant, recombinant vector as well as preparation method and application of Escherichia coli membrane protein ZraS mutant
  • Escherichia coli membrane protein ZraS mutant, gene for coding mutant, recombinant vector as well as preparation method and application of Escherichia coli membrane protein ZraS mutant

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preparation example Construction

[0076] The present invention also provides a preparation method for the above-mentioned mutant, comprising: constructing SP-ZraS phage; adopting phage-assisted continuous directed evolution (PACE) to alternately evolve the substrate specificity of SP-ZraS phage to lead ions by positive screening and negative screening , to obtain an evolutionary sample; carry out amino acid sequencing analysis on the evolutionary sample to obtain mutation site information; construct the expression vector of the membrane protein ZraS mutant according to the mutation site information, denote as pZraS, and express to obtain the membrane protein ZraS mutant .

[0077] In the present invention, the preparation method specifically includes:

[0078] 1.1 Construction of positive screening helper plasmid and host

[0079] 1.11 Construction of the gIII protein expression plasmid AP1: the gIII protein is initiated by the promoter psp, and the downstream of the promoter psp is inserted into the recognit...

Embodiment 1

[0102] The ZraS mutation site obtained by directed evolution for lead ion substrate recognition specificity

[0103] 1. Construction of PACE directed evolution helper plasmid

[0104] 1.1 Construction of positive screening helper plasmid and host

[0105] 1) Construction of the gIII protein expression plasmid AP1: the gIII protein is activated by the promoter psp, and the recognition site nucleic acid sequence of the ZraR regulatory protein is inserted downstream of the promoter. AP1 map see image 3 .

[0106] 2) Construction of regulatory protein ZraR and mutagenic gene expression plasmid MP-ZraR: the vector template is mutagenic plasmid MP4, and the ZraR gene sequence is inserted upstream of the arabinose promoter of mutagenic plasmid MP4, and the promoter is J23109. The plasmid can express the regulatory protein ZraR and provide a higher mutation rate under the induction of arabinose during DNA replication. MP-ZraR map see Figure 4 .

[0107] 3) Co-transform E.coli ...

Embodiment 2

[0134] Construction of engineering strains

[0135] 1. Select the mutation sites or combinations obtained in Example 1 to construct different ZraS mutant expression vectors pZraS, and the promoter of ZraS is the endogenous promoter ZraSp.

[0136] 2. Construct the expression vector pLuxAB-ZraR of the reporter gene luxAB and the regulatory protein ZraR, see the plasmid map figure 2 . The reporter gene luxAB and the regulatory protein ZraR are expressed in tandem, and are activated by the promoter zraPp, and two binding sites of the regulatory protein ZraR are set upstream of the promoter.

[0137] 3. Different pZraS vectors were co-transformed with pLuxAB-ZraR plasmids to E. coli S1030 competent cells to obtain different recombinant mutant strains.

[0138] The expression host used in the present invention is Escherichia coli S1030 carrying the F plasmid, derived from E.coli DH10B, provided by David R Liu's laboratory, and its genetic information has been reported in relevan...

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Abstract

The invention provides an Escherichia coli bi-component regulation and control system membrane protein ZraS mutant, a gene for coding the mutant, a recombinant vector, a preparation method and an application of the Escherichia coli bi-component regulation and control system membrane protein ZraS mutant. The mutant comprises one or more of S154T, A214S, Q313R and T415A, and the mutant comprises one or more of S154T, A214S, Q313R and T415A. The preparation method of the mutants comprises the following steps: constructing the SP-ZraS bacteriophage; alternately evolving the substrate specificity of the SP-ZraS bacteriophage on the lead ions through positive screening and negative screening by adopting a bacteriophage-assisted continuous directed evolution (PACE) method, so as to obtain an evolutionary sample; performing amino acid sequencing analysis on the evolutionary sample to obtain mutation site information; and constructing an expression vector of the membrane protein ZraS mutant according to mutation site information, recording the expression vector as pZraS, and performing expression to obtain the membrane protein ZraS mutant. The response sensitivity of the mutant to metal lead ions is remarkably improved compared with that of a wild type system, the regulation and control range to lead is enlarged, and the detection limit is as low as 1 mu M; meanwhile, when the ion concentration range is 0-10 mu M, the specific and sensitive detection on the heavy metal lead ions can be realized.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an Escherichia coli membrane protein ZraS mutant, a gene encoding the mutant, a recombinant vector, a preparation method and an application. Background technique [0002] Lead is an environmental heavy metal pollutant that is widely distributed in nature. There is a certain amount of lead in soil, air and water to varying degrees. These lead will enter our food through air, water and soil. Lead in food is mainly brought into food raw materials by environmental pollution, and then absorbed into the body through diet. Therefore, the lead in food exceeds the standard, mostly because the production company does not strictly control the raw materials and uses raw materials with excessive lead content. Of course, the possibility of migration from production equipment into food is not ruled out. Lead has a long half-life in the body, so it can accumulate in the ...

Claims

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Application Information

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IPC IPC(8): C07K14/245C12N15/31C12N15/70C12N1/21C12Q1/10C12R1/19
CPCC07K14/245C12N15/70C12Q1/10
Inventor 刘复荣张润钊崔金明李小明
Owner GUANGZHOU INST OF ADVANCED TECH CHINESE ACAD OF SCI
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