This article describes a method for generating double crossover recombinants in Pseudomonas aeruginosa using a non-replicative plasmid. The process involves antibiotic selection and sucrose sensitivity to confirm successful genetic modifications.
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-warmed PIA plates, supplemented with 10% sucrose. Then incubate the plates overnight at 37 degrees Celsius.
On the next day, remove the plates from the incubator and inspect them for growth. The sucrose resistant colony should be double crossover recombinants. Use sterile toothpicks to patch at least 20 colonies onto pre-warmed plates of PIA, PIA supplemented with 10% sucrose, and PIA supplemented with carbenicillin.
Incubate the plates overnight and examine them for growth on the next day. True double crossover recombinants will be carbenicillin-sensitive and sucrose-resistant.
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