Development of a Method for Simultaneous Generation of Multiple Genetic Modification in Serovar Typhimurium

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.