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 λ 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 the Salmonella enterica serovar Typhimurium chromosome. The method combines an efficient seamless assembly system in vitro, red homologous recombination in vivo, and counterselection marker sacB. To test this method, with the seamless assembly system, various modification fragments for target genes cpxR, cpxA, and acrB were rapidly and efficiently constructed in vitro. sacBKan cassettes generated via polymerase chain reaction were inserted into the target loci in the genome of Salmonella Typhimurium 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 the sacBKan cassettes in the chromosomes of the intermediate strains. Using this method, we successfully carried out site-directed mutagenesis, seamless deletion, and 3 × FLAG tagging of the target genes. This method can be used in any bacterial species that supports sacB gene activity and λ red-mediated recombination, allowing in-depth functional analysis of bacterial genes.

为了全面分析细菌基因功能，重要的是同时在靶基因内产生多种遗传修饰。然而，当前的基因工程方法主要使用基于自杀载体或λRed同源重组的系统，既繁琐又在技术上难以执行。在这里，我们开发了一种灵活简便的方法，可以在同一位置的同一位点上同时构建多个修饰肠沙门氏菌血清鼠伤寒染色体。该方法结合了高效的无缝装配系统体外，红色同源重组 体内和反选择标记 沙巴。为了测试此方法，通过无缝装配系统，对靶基因进行了各种修饰cpxR， cpxA和 acrB 快速有效地建造 体外。 萨班 通过聚合酶链反应产生的盒被插入到基因组的靶基因座中。 沙门氏菌鼠伤寒菌株CVCC541。选择所得的含pKD46的卡那霉素抗性重组体，并将其用作中间菌株。然后使用各种同源重组DNA片段通过等位基因交换同时进行多个靶基因修饰，以取代萨班中间菌株的染色体中的盒。使用这种方法，我们成功地进行了目标基因的定点诱变，无缝缺失和3×FLAG标记。此方法可用于支持沙巴 基因活性和λ红色介导的重组，可对细菌基因进行深入的功能分析。