Generating mutant strains is an essential component of microbial genetics. Natural genetic transformation, a process for the uptake and integration of foreign DNA, is shared by diverse microbial species and can be exploited for making mutant strains. Canonically, this process has been used to generate single mutants and sequentially to generate strains with multiple mutations. Recently, we have described a method for multiplex genome editing by natural transformation (MuGENT), which allows the generation of strains with multiple scarless mutations in a single step. Here, we provide a detailed description of the methods used for mutagenesis of the cholera pathogen Vibrio cholerae with a particular emphasis on mutagenesis via MuGENT.
Keywords: Accelerated evolution; Combinatorial mutagenesis; Cotransformation; Multiplex genome editing; Natural transformation; Vibrio cholerae.