Jing Wenxian, Liu Juan, Wu Shanshan, Chen Qiwei, Li Xuerui, Liu Yongsheng
State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China.
Front Genet. 2020 Sep 24;11:563491. doi: 10.3389/fgene.2020.563491. eCollection 2020.
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 serovar Typhimurium chromosome. The method combines an efficient seamless assembly system , red homologous recombination , and counterselection marker . To test this method, with the seamless assembly system, various modification fragments for target genes , , and were rapidly and efficiently constructed . cassettes generated via polymerase chain reaction were inserted into the target loci in the genome of 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 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 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，在目标基因内同时产生多种基因修饰非常重要。然而，目前的基因工程方法主要使用基于自杀载体或λ红同源重组的系统，操作繁琐且技术难度大。在此，我们开发了一种灵活简便的方法，可在鼠伤寒沙门氏菌染色体的同一基因座上同时构建多种修饰。该方法结合了高效无缝组装系统、红同源重组和反选择标记。为了测试该方法，利用无缝组装系统，快速高效地构建了针对目标基因、和的各种修饰片段。通过聚合酶链反应产生的盒式片段被插入鼠伤寒沙门氏菌CVCC541菌株基因组的目标位点。筛选出产生的含pKD46的卡那霉素抗性重组体并用作中间菌株。然后使用各种同源重组DNA片段通过等位基因交换同时对多个目标基因进行修饰，以取代中间菌株染色体中的盒式片段。利用该方法，我们成功地对目标基因进行了定点诱变、无缝缺失和3×FLAG标记。该方法可用于任何支持基因活性和λ红介导重组的细菌物种，从而对细菌基因进行深入的功能分析。
