Isolation of Gram-Negative Bacteria with Genomic Transposon Insertions

Begin by thawing a frozen culture of the mated donor and recipient Gram-negative bacterial population on ice.

The recipient cells carry transposons, short DNA segments that include a resistance gene for the antibiotic kanamycin. These transposons are randomly integrated into the host genome.

Add the culture to a nutrient-rich agar plate containing kanamycin but without donor-specific nutrients.

Add sterile beads and gently rotate the plate to spread the culture uniformly.

Remove the beads and incubate. Recipient bacteria utilize nutrients, but only those with integrated transposons resist kanamycin, leading to colony formation.

The donor and recipient bacteria without transposons fail to grow.

Count colony-forming units to estimate the number of recipient bacteria with unique transposon insertions.

Add fresh media and collect the bacterial population.

Centrifuge to pellet the bacteria and discard the supernatant.

Resuspend the pellet in media with cryoprotectant to preserve bacterial viability.

Thaw an aliquot of the frozen mating on ice. Then, use sterile glass beads to spread 150 microliters of the dilution, onto each 30 by 150 millimeter Luria-Bertani agar plate, supplemented with kanamyc