Natural Cotransformation and Multiplex Genome Editing by Natural Transformation (MuGENT) of Vibrio cholerae
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.
Key wordsVibrio cholerae Natural transformation Multiplex genome editing Combinatorial mutagenesis Accelerated evolution Cotransformation
- 4.Gulig PA, Tucker MS, Thiaville PC, Joseph JL, Brown RN (2009) USER friendly cloning coupled with chitin-based natural transformation enables rapid mutagenesis of Vibrio vulnificus. Appl Environ Microbiol 75(15):4936–4949. Epub 2009/06/09. https://doi.org/10.1128/AEM.02564-08 CrossRefPubMedPubMedCentralGoogle Scholar
- 6.Blokesch M (2012) TransFLP—a method to genetically modify Vibrio cholerae based on natural transformation and FLP-recombination. J Vis Exp 68. Epub 2012/10/25. https://doi.org/10.3791/3761