Assessing the Role of Gene Copy Number in the Evolution of Bacterial Antibiotic Resistance

Begin with cultures of three E. coli strains —one lacking the ampicillin resistance gene, one carrying a single chromosomal copy of the resistance gene, and one harboring the resistance gene on a multicopy plasmid.

Transfer aliquots from each culture into fresh media containing a subinhibitory concentration of the antibiotic ceftazidime.

Incubate the cultures to allow spontaneous mutations in the ampicillin resistance gene that may confer cross-resistance to ceftazidime.

Post-incubation, measure optical density to assess bacterial survival.

Repeat this process daily by transferring aliquots into fresh media, doubling the ceftazidime concentration each time.

Incubate and measure optical density after each transfer to monitor survival under increasing antibiotic pressure.

The strain harboring the multicopy plasmid alone survives at higher ceftazidime levels, as increased gene copies raise the likelihood of acquiring resistance mutations.

This demonstrates that higher gene copy number enhances the evolution of bacterial antibiotic resistance.

First, streak the MG1655 strains of interest on the LB agar plates. Then add 200 microliters of LB medium in each well of a 96-well plate.

Next, inoculat