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Method for preparing gram-negative bacterium competent cells through outer membrane defects

A gram-negative, competent cell technology, applied in the field of microbial genetic engineering, can solve problems such as hindering the transfer of DNA molecules in competent cells, and achieve the effect of improving transformation efficiency and convenient operation.

Active Publication Date: 2020-03-13
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, it is likely that LPS on the outer membrane hinders the transfer of DNA molecules in competent cells

Method used

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  • Method for preparing gram-negative bacterium competent cells through outer membrane defects
  • Method for preparing gram-negative bacterium competent cells through outer membrane defects
  • Method for preparing gram-negative bacterium competent cells through outer membrane defects

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1. Preparation of electroporation competent cells of Halomonas by knocking out outer membrane lipid A synthesis and modification related genes

[0058] After knocking out the genes related to outer membrane lipid A synthesis and modification in the genome of Halomonas bluephagenesis TD01, an LPS-deficient strain was obtained, which can become electroporation-competent cells after being treated with sucrose-glycerol buffer Plasmid containing the spectinomycin resistance gene.

[0059] The specific operation process is as follows:

[0060] Step 1. Use CRISPR-Cas9 technology to knock out the genes LpxA, LpxC, kdtA, LpxL, and LpxM related to the synthesis and modification of outer membrane lipid A in the genome of Halomonas bluephagenesis TD01, and obtain the outer membrane-deficient strain of the halophilic bacteria . details as follows:

[0061] 1. Knock out genes

[0062] The original expression vector used to express sgRNA and donor DNA is the pSEVA241 plasm...

Embodiment 2

[0080] Example 2. Preparation of electroporation competent cells of Halomonas by knocking out outer membrane lipid A synthesis and modification related genes, and transformation of plasmid containing gfp gene

[0081] Using Halomonas (H. bluephagenesis) TD01 as the starting strain, after knocking out lipid A synthesis and modifying related genes, the LPS-deficient strain of the strain was obtained, and the strain could become electroporation competent after being treated with sucrose buffer Cells can be electroporated with plasmids containing the spectinomycin resistance gene and the gfp gene.

[0082] The specific operation process is as follows:

[0083] Step 1. Use CRISPR-Cas9 technology to knock out the outer membrane lipid A synthesis and modification related genes LpxA, LpxC, KdtA, LpxL, and LpxM in the genome of H. bluephagenesis TD01 to obtain the outer membrane of H. bluephagenesis Deficient strains. The specific operation is the same as step one of embodiment 1.

...

Embodiment 3

[0090] Example 3. Preparation of Halomonas Electrotransfer Competent Cells by Inhibiting Outer Membrane LPS Synthesis and Modifying Related Genes

[0091] Halomonas campaniensis (Halomonas campaniensis) LS21 is a Gram-negative halophilic bacterium screened by our laboratory, which has a very good prospect for industrial production. After inhibiting the outer membrane LPS synthesis and modifying related genes, the LPS-deficient strain of the strain was obtained, and the strain could become electroporation competent cells after being treated with sucrose-glycerol buffer, and the plasmid containing the chloramphenicol resistance gene could be electroporated.

[0092] The specific operation process is as follows:

[0093] Step 1. Use sRNAi technology to inhibit the outer membrane LPS synthesis and modification related genes in the genome of H. campaniensis LS21, including MsbA, WzX, WzY, WaaL, WaaF, and WaaC, and obtain the outer membrane-defective type of H. campaniensis strain....

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Abstract

The invention discloses a method for preparing gram-negative bacterium competent cells through outer membrane defects. The method comprises the following steps: knocking out and / or inhibiting genes related to outer membrane lipopolysaccharide (LPS) in a genome of gram-negative bacteria to obtain an outer membrane defect type strain; and preparing competent cells by utilizing the outer membrane defect type strain. The method provided by the invention can improve the conversion efficiency of wild-type strains (such as pseudomonas insecticola) which can be subjected to electrotransformation or chemical transformation. More importantly, for wild type gram-negative strains (such as halophilic bacteria and the like) which cannot be electrotransformed or chemically transformed, the competent cells prepared from the membrane-deficient strains can be electrotransformed or chemically transformed into DNA molecules, can be transformed into various plasmids, and express functional genes of the plasmids. The method plays an important role in gene editing of gram-negative bacteria.

Description

technical field [0001] The invention relates to the technical field of microbial genetic engineering, in particular to a method for preparing Gram-negative bacterial competent cells through outer membrane defects. Background technique [0002] In the natural state, some bacteria can transfer plasmid DNA into new hosts through conjugation. Cohen was the first to successfully transform plasmid DNA molecules into CaCl in 1972. 2 Competent cells of Gram-negative bacteria Escherichia coli produced by the law (Cohen, S.N., et al. (1972). "Nonchromosomal antibiotic resistance inbacteria: genetic transformation of Escherichia coli by R-factor DNA." ProcNatl Acad Sci U S A 69(8):2110-2114), since then, the preparation and transformation of competent cells has become an important technology in modern molecular biology experiments, and has played an important role in gene editing and DNA library construction. The commonly used transformation methods are calcium chloride method, Hanah...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/87C12R1/01C12R1/19C12R1/38
CPCC07K14/195C12N15/87
Inventor 陈国强王子瑜秦琴
Owner TSINGHUA UNIV
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