Transposon-based random mutagenesis of bacterial genomes has proven to be a powerful genetic tool for the identification of genes and regulatory elements that contribute to specific phenotypes. One such approach that has been used in Vibrio cholerae for many years is the introduction of mariner transposons to generate random libraries of mutants. These libraries have been successfully used for a wide variety of genetic screens and selections in this important bacterial pathogen. Here we present a detailed protocol for the use of plasmid pFD1 (containing the mariner transposon magellan3) to create mutant libraries in V. cholerae.
Keywords: Mariner transposon; Nested PCR; Random mutagenesis; Random-primed sequencing; Transposon mutagenesis.