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, inoculate 48 isolated colonies of each strain in individual wells of the plate. After inoculation, incubate the plate overnight at 37 degrees Celsius on a shaker at 200 revolutions per minute. Again, inoculate two microliters of the culture from each well with the founder population to a new 96-well plate with 198 microliters of LB medium in individual wells.

This will start the evolutionary rescue experiment. Incubate the plate at 37 degrees Celsius on a shaker at 200 revolutions per minute for 20 hours. Each day, double the concentration of the antibiotic from that of the previous day.

The next day, transfer 2 microliters of the respective culture to a fresh 96-well plate and continue this every day. Record the optical density reading at 600 nanometers from the individual wells on a plate reader.

A critical step of the protocol is the experimental evolution and their increasing concentrations of antibiotics. The rate of change of antibiotic concentrations is one of the parameters that can be modified to promote the evolution of antibiotic resistance.

Again, incubate the plate at 37 degrees Celsius on a shaker at 200 revolutions per minute for 20 hours. Then measure the optical density reading at 600 nanometers for each surviving population every day on the plate reader.