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Chemical conversion mediation based escherichia coli gene knockout method by CRISPR/Cas9 system

A technology of chemical transformation of Escherichia coli, which is applied in the field of gene knockout, can solve the problem of high cost and achieve the effect of simple operation

Inactive Publication Date: 2018-01-30
ZHEJIANG UNIV OF TECH
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Problems solved by technology

[0005] The existing CRISPR technology knockout Escherichia coli gene knockout system combines Cas9 protein and λ-Red homologous recombination system to realize target gene knockout. The transformation method introduces exogenous DNA into host cells, and has high requirements on the purity of DNA fragments and the preparation of competent cells.
Electrotransformation requires special electrotransformation equipment (electrotransfer instrument) and expensive experimental consumables (electric shock cup), so it is not suitable for carrying out a large number of single gene knockout work at the same time

Method used

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  • Chemical conversion mediation based escherichia coli gene knockout method by CRISPR/Cas9 system
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  • Chemical conversion mediation based escherichia coli gene knockout method by CRISPR/Cas9 system

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Embodiment 1

[0037] Knockout of panD gene in Escherichia coli BL21 in embodiment 1

[0038] 1. Transfect the pCas9 plasmid calcium into E. coli BL21, which is denoted as E. coli BL21pCas9, including the following steps:

[0039] (1) Competent preparation of Escherichia coli BL21: Pick a single colony of Escherichia coli BL21 and inoculate it in a 5mL LB liquid test tube, cultivate it at 37°C and 200r / min until OD 600 = 0.6, centrifuged at low temperature (4°C), the obtained cells were washed with solution A (purchased from TaKaRa Competent Cell Preparation Kit) and solution B (purchased from TaKaRa Competent Cell Preparation Kit) respectively, and then resuspended in solution B, the chemically transformed competent cells of Escherichia coli BL21 were obtained.

[0040] (2) Transformation: Mix 5 μL of pCas9 plasmid and 50 μL of E. coli BL21 competent cells, and put them in an ice bath for 30 minutes. After heat-shocking the mixture at 42°C for 90 seconds, place it on ice for 1-2 minutes, a...

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Abstract

The invention discloses a chemical conversion mediation based escherichia coli gene knockout method by a CRISPR / Cas9 system. A chemical conversion method is adopted to guide foreign DNA into a host cell, and escherichia coli gene knockout is realized successfully by a gamma-Red homologous recombination system and the CRISPR / Cas9 system which are pre-guided into the host cell. Compared with existing escherichia coli gene knockout methods, the chemical conversion mediation based escherichia coli gene knockout method has the advantage that efficient and accurate target gene knockout can be realized in escherichia coli. The intra-bacteria gene knockout method is simple, convenient and rapid to operate, has no special requirements on experimental devices, has low experimental cost, and is particularly applicable to knockout of high-throughput genes.

Description

(1) Technical field [0001] The invention relates to the technical field of gene knockout, in particular to a method for specifically knocking out a chromosomal target gene in Escherichia coli based on a CRISPR / Cas9 system mediated by chemical transformation. (2) Background technology [0002] Escherichia coli (Escherichia coli) is widely used in genetic engineering, genetic engineering, fermentation engineering, metabolic engineering and other fields, and has broad application prospects. Gene knockout involves the introduction of exogenous DNA and its homologous recombination with chromosomal DNA. Datsenko and Wanner greatly improved the recombination efficiency of E. coli by introducing the λ-Red homologous recombination system, and invented a one-step knockout E. coli chromosomal gene technology (Datsenko & Wanner, PNAS, 2000, 97:6640-6645). The CRISPR system can cause double-strand breaks in the corresponding region of the target gene, and the combination of the λ-Red ho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/90C12N1/21C12R1/19
Inventor 孙东昌裘娟萍王琳
Owner ZHEJIANG UNIV OF TECH
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