Method of performing traceless knockout and integration on gene of Acidithiobacillus caldus

Abstract

The invention discloses a method of performing traceless knockout on a chromogene or inserting an exogenous gene in a chromosome of Acidithiobacillus caldus. The method comprises the following steps: transferring a homologous recombinant plasmid containing plentiful restriction enzyme digestion sites of endonuclease I-SceI into the Acidithiobacillus caldus in a conjugation transfer manner, simultaneously, directionally inserting the homologous recombinant plasmid into the chromosome of Acidithiobacillus caldus by use of a homologous recombinant system of a cell itself, transferring a screened single crossover to an I-SceI expression plasmid by an electroporation method, performing secondary homologous recombination on a single recon chromosome under a stress that the I-SceI enzyme cuts the chromosome, and optimizing and screening a double crossover bacterial strain to realize knockout and insertion of the gene. The invention also constructs a suicide type plasmid vector pSDUDI for homologous recombination and a plasmid pSDU1-I-SceI for expressing the I-SceI enzyme effectively, and provides convenience for the traceless knockout and the integration of the chromogene in the Acidithiobacillus caldus. The method provided by the invention provides a new way for intensive study on and modification of Acidithiobacillus caldus.

Description

technical field

[0001] The invention relates to a gene traceless knockout and integration method of acidophilic Thiobacillus acidothermophilus, in particular to a method for seamlessly knocking out the chromosome gene of acidophilic Thiobacillus thermophilic or inserting an exogenous gene into the chromosome of acidophilus Thiobacillus thermophilic method, and a suicide plasmid vector pSDUDI for homologous recombination in chromosomal gene scarless knockout and integration, and a plasmid pSDU1-I- expressing meganuclease in Thiobacillus acidophilus. SceI. Background technique

[0002] Acidithiobacillus caduls (A.caldus) is a kind of Gram-negative obligate autotrophic sulfur-oxidizing bacteria, which plays a very important role in bacterial leaching and can significantly increase the iron-oxidizing bacteria such as Acidithiobacillus The leaching efficiency of ferroxidans, Leptospirillum ferrooxidans, etc. In recent years, studies have found that A. can improve the ore leachi...

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