This article describes a method for creating a transposon library in Gram-negative bacteria. The process involves the use of recipient cells with integrated transposons that confer resistance to kanamycin, allowing for the selection of successful integrations.
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 kanamycin.
Dispose of the used tube containing excess mating, and incubate plates at 37 degrees Celsius for 14 hours. After the incubation count the colony forming units on each plate to estimate the total mutants in the transposon library. Count 20%of at least three plates to determine the colony count estimate for the entire group of plates.
After calculating the estimated colony yield, add three to five milliliters of LB to each plate, and scrape off the bacteria using a sterile scraping tool. Pull bacterial suspensions from all plates into 50 milliliter conical tubes, and pellet the suspensions by centrifuging at 5, 000 times G, for seven minutes. Discard the supernatant and re-suspend the pellet in five milliliters of LB, supplemented with 30% glycerol.
Then, make one milliliter aliquots of the transposon library in cryovials and store them at minus 80 degrees Celsius.
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