Generating Double-Crossover Recombinants of <i>Pseudomonas aeruginosa</i> for Targeted Gene Deletion

Source: Valentine, M. E., et al. Generation of In-Frame Gene Deletion Mutants in Pseudomonas aeruginosa and Testing for Virulence Attenuation in a Simple Mouse Model of Infection. J. Vis. Exp. (2020).This video demonstrates the generation of double-crossover recombinants in Pseudomonas aeruginosa using a plasmid-based gene deletion strategy. The process involves selecting bacteria that harbor a non-replicative plasmid integrated into the genome via a single crossover and then inducing a second recombination event under sucrose selection to excise the plasmid. Successful double-crossover recombination is confirmed by the loss of antibiotic resistance and the ability to grow on sucrose-containing media.