A method for realizing scarless homologous recombination of Escherichia coli genes

A technology of Escherichia coli and homologous recombination, applied in recombinant DNA technology, biochemical equipment and methods, viruses/phages, etc., can solve problems such as the negative impact of accuracy and recombination efficiency

Inactive Publication Date: 2019-01-11
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Residual site sequences after such homologous recombination will have a serious negative impact on the accuracy and recombination efficiency of subsequent gene homologous recombination operations

Method used

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  • A method for realizing scarless homologous recombination of Escherichia coli genes
  • A method for realizing scarless homologous recombination of Escherichia coli genes
  • A method for realizing scarless homologous recombination of Escherichia coli genes

Examples

Experimental program
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Effect test

Embodiment 1

[0022] Example 1 Construct the kan operon of the kanamycin resistance gene initiated by the lac operator sequence, and replace the wild-type lacI gene and its promoter of Escherichia coli DH5α by homologous recombination. The recombinant plasmid pUC19-cat-PlacIq-lacI composed of a traceless recombination tool gene string composed of the lacI repressor gene in reverse, uses the cat-PlacIq-lacI tool gene string to replace the natural promoter of the talB gene in E. coli DH5α with trc promoter, the steps are as follows:

[0023] 1.1. The plasmid pKD46 was electrotransformed into Escherichia coli DH5α, and the strain that successfully introduced the plasmid was obtained by screening with ampicillin plate.

[0024] 1.2. Use the upstream and downstream primers lacI-Plac-kan-F and lacI-kan-R (DNA sequences shown in SEQ ID NO.1 and SEQ ID NO.2 respectively) to obtain Plac-lacO- For the kan operon gene, its homologous recombination was used to replace the wild-type lacI gene and its p...

Embodiment 2

[0030] Example 2 Construct the kan operon of the kanamycin resistance gene initiated by the lac operator sequence, and replace the wild-type lacI gene and its promoter of Escherichia coli BL21 (DE3) by homologous recombination, and construct a The recombinant plasmid pUC19-cat-PlacIq-lacI, which is composed of a traceless recombination tool gene string composed of cat and lacI repressor gene in reverse, uses the cat-PlacIq-lacI tool gene string to integrate the talB gene in Escherichia coli BL21 (DE3) Replace the natural promoter with the trc promoter, the steps are as follows:

[0031] 2.1. The plasmid pKD46 was electrotransformed into Escherichia coli BL21 (DE3), and the strains successfully introduced with the plasmid were obtained by screening with ampicillin plate.

[0032] 2.2. Use the upstream and downstream primers lacI-Plac-kan-F and lacI-kan-R (DNA sequences shown in SEQ ID NO.1 and SEQ ID NO.2 respectively) to obtain Plac-lacO- from plasmid pKD4 by pcr amplification...

Embodiment 3

[0038] Example 3 Construct the kan operon of the kanamycin resistance gene initiated by the lac operator sequence, and replace the wild-type lacI gene and its promoter of Escherichia coli JM109 by homologous recombination. The recombinant plasmid pUC19-tet-PlacIq-lacI, which is a seamless recombination tool gene string composed of a reverse connection of the target gene, uses the tet-PlacIq-lacI tool gene string to replace the natural promoter of the talB gene in Escherichia coli JM109 with the trc promoter ,Proceed as follows:

[0039] 3.1. The plasmid pKD46 was electrotransformed into Escherichia coli JM109, and the strains successfully introduced with the plasmid were obtained by screening with ampicillin plate.

[0040] 3.2. Use the upstream and downstream primers lacI-Plac-kan-F and lacI-kan-R (DNA sequences shown in SEQ ID NO.1 and SEQ ID NO.2 respectively) to obtain Plac-lacO- from plasmid pKD4 by pcr amplification For the kan operon gene, its homologous recombination ...

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Abstract

The invention discloses a method for implementing Escherichia coli gene traceless homologous recombination, which is implemented on the basis of a Lac repressible system and resistance screening. An Escherichia coli recombinant bacterium comprising a resistance operon controlled by Lac operator sequence and a traceless recombination tool gene cluster comprising positive and negative screening markers are constructed to establish a method for implementing Escherichia coli gene traceless homologous recombination, thereby achieving the goal of performing traceless recombination on the target site on the Escherichia coli chromosome by the target gene, and having important meanings for doing gene editing research work on Escherichia coli chromosome.

Description

technical field [0001] The invention belongs to the field of biochemical engineering, and in particular relates to a method for realizing traceless homologous recombination of Escherichia coli genes based on a Lac repression system and resistance screening. Background technique [0002] Escherichia coli has the advantages of relatively simple genetic manipulation, fast growth, and low fermentation cost, and is currently one of the most commonly used model strains in genetic engineering. The core technology of E. coli metabolic engineering is the mutation, deletion, insertion or replacement of genes in the E. coli genome. Red homologous recombination technology is a gene targeting technology that can be genetically manipulated at the chromosome level. It has the characteristics of short homologous sequences, wide range of applications and flexible operation strategies. It is currently the most commonly used homologous recombination in E. coli technology. However, after edit...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/72
CPCC12N15/72C12N2800/101
Inventor 黄磊姜灵轩徐志南蔡谨
Owner ZHEJIANG UNIV
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