Generating Double-Crossover Recombinants of Pseudomonas aeruginosa for Targeted Gene Deletion

Begin with a culture of recombinant Pseudomonas aeruginosa in an antibiotic selection medium.

These bacteria harbor a non-replicative plasmid integrated into the genome near the bacterial target gene intended for deletion.

Integration occurred via homologous sequences shared between the plasmid and genome, resulting in a single crossover recombination.

The plasmid encodes an antibiotic resistance gene and the sacB gene, which confers sucrose sensitivity.

Streak the bacteria onto a medium containing sucrose but lacking the antibiotic and incubate.

Without antibiotic pressure, a second recombination, or a double crossover, occurs between homologous regions, excising the plasmid and target gene.

During bacterial multiplication, the excised plasmid doesn't get passed on, making the bacteria sucrose-tolerant.

Patch the bacteria onto a non-selective medium and a medium containing sucrose or the antibiotic, and incubate.

Growth on the non-selective and sucrose media, but not on the antibiotic medium, confirms a successful double crossover.

Start by growing single crossover, recombinant colonies in Pseudomonas Isolation Broth, or PIB. Inoculate and streak 10 microliters of each culture onto pre-wa