Development of a Method for Simultaneous Generation of Multiple Genetic Modification inSalmonella entericaSerovar Typhimurium

文献类型： 外文期刊

第一作者： Jing, Wenxian

作者： Jing, Wenxian;Liu, Juan;Wu, Shanshan;Chen, Qiwei;Li, Xuerui;Liu, Yongsheng

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关键词： gene modification; simultaneous construction; seamless assembly system; red homologous recombination; Salmonella entericaserovar Typhimurium

期刊名称：FRONTIERS IN GENETICS （ 影响因子：4.599； 五年影响因子：4.888 ）

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年卷期： 2020 年 11 卷

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收录情况： SCI

摘要： To comprehensively analyze bacterial gene function, it is important to simultaneously generate multiple genetic modifications within the target gene. However, current genetic engineering approaches, which mainly use suicide vector- or lambda red homologous recombination-based systems, are tedious and technically difficult to perform. Here, we developed a flexible and easy method to simultaneously construct multiple modifications at the same locus on theSalmonella entericaserovar Typhimurium chromosome. The method combines an efficient seamless assembly systemin vitro, red homologous recombinationin vivo, and counterselection markersacB. To test this method, with the seamless assembly system, various modification fragments for target genescpxR,cpxA, andacrBwere rapidly and efficiently constructedin vitro.sacBKancassettes generated via polymerase chain reaction were inserted into the target loci in the genome ofSalmonellaTyphimurium strain CVCC541. The resulting pKD46-containing kanamycin-resistant recombinants were selected and used as intermediate strains. Multiple target gene modifications were then carried out simultaneously via allelic exchange using various homologous recombinogenic DNA fragments to replace thesacBKancassettes in the chromosomes of the intermediate strains. Using this method, we successfully carried out site-directed mutagenesis, seamless deletion, and 3 x FLAG tagging of the target genes. This method can be used in any bacterial species that supportssacBgene activity and lambda red-mediated recombination, allowing in-depth functional analysis of bacterial genes.

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