The overall goal of this experimental method is to test the role of multicopy plasmids in the evolution of antibiotic resistance. This method can help to answer key questions in the field of microbiology such as the role of multicopy plasmids in the evolution of bacterial innovations. The main advantage of this technique is that it only requires basic molecular biology methods and a simple experimental design.
It's really cool. First, construct the e coli MG1655 strain that will carry the antibody resistance gene in the chromosome in the lambda phage in equation side ATTB. To do so, PCR amplify the 500 base paralong regions on both sides of the ATTB site.
Then PCR amplify the antibiotic resistance gene. Then use isothermal assembly at 50 degrees Celsius for 30 minutes to fuse the homology regions to the PCR product. After isothermal assembly, transfer one microliter of the product to 40 microliters of electrocompetent MG1655 cells into a two millimeter cuvette.
Then electroporate the cells at 4 degrees Celsius and 2.5 kilovolts. After electroporation, transfer the cells in one milliliter of LB broth with 0.2%arabinose. Then pipette the LB broth containing cells to a microfuge tube.
Shake the tube intensely for two hours at 30 degrees Celsius. After two hours, plate 100 microliters of the culture on the LB agar plate, supplemented with antibiotics. Then spin down the remaining cells.
And re-suspend the pellet in 100 microliters of fresh LB broth. Re-plate these cells on another LB agar plate. Then incubate the plates at 42 degrees Celsius overnight.
The following day, PCR amplify the clones to verify the construct using external primers ybhC extern and ybhB extern. Then confirm the amplicon size through the agarose shell electrophoresis. Parallelly, conduct DNA sequencing to verify the sequence of the inserted gene.
To construct the strain to carry the antibody resistance gene in the multicopy plasmid, first PCR amplify the antibiotic resistance gene blaTEM-1. After the PCR is completed, phosphorylate the amplified product using T4 polynucleotide kinase. Next, use high-fidelity polymerase and oligonucleotides PBAD FINV and PBAD RINV, to PCR amplify the PBAD plasmid backbone.
Then ligate the two PCR fragments using T4 ligase. Next, electroporate 40 microliters DH5 alpha cells with 1 microliter of the ligation product. Then select the appropriate antibiotic for the antibiotic resistance gene and the plasmid backbone on agar plates.
Then perform plasmid extraction using a DNA extraction kit, following the manufacturer's instructions. Then perform sanger sequencing of the resistance gene of interest to confirm the absence of any possible mutations before the evolution experiments. After verification of the sequence, electroporate the plasmid in the e coli strain MG1655 to finally obtain the plasmid carrying the MG1655 strain.
The most critical steps in this protocol are the construction of the model systems strains and the reconstruction of the mutations observed during experimental evolution. 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 degree 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 fresh 96 well plate and continue this everyday. 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 everyday on the plate reader.
Freeze the populations periodically, every three to five days. Extract the total DNA from the evolved populations and the clones from different strains and treatments. Next, measure the absorbance ratios at 260:280, and 260:230 nanometers on the spectrophotometer to determine the quality of the DNA.
Then use a fluorescent DNA binding dye and confirm the concentration of the DNA by measuring the fluorescence on the plate reader. Additionally, subject the DNA to agarose gel electrophoresis to confirm that there is no DNA degradation or RNA contamination. Then, perform deep DNA sequencing with samples obtained from whole populations and individual clones to study the genetic basis of antibiotic resistance.
This study is an example of the possible outcomes of the experimental system using three e coli strains. These strains are e coli MG1655, MG1655 carrying the resistance gene in the chromosome MG1655 resA and MG1655 carrying the resistance gene in the multicopy plasmid, MG1655 pRESA. The antibiotics used are ceftazidime and meropenem.
This left panel shows the survival curves under increasing concentrations of ceftazidime. From the plot, it is clear that the subset of population of strain MG1655 pRESA are able to grow beyond 14 days even after doubling the ceftazidime concentration each day. On the contrary, the other strains do not survive beyond 8 days when exposed to similar concentrations of ceftazidime.
However, the same strains do not show significant differences in the survival period when exposed to similar concentration of another antibiotic, meropenem. All the three strains survive only between 6 to 8 days when exposed to meropenem. This indicates that the presence of the gene resA in the multicopy plasmid potentiates the evolution of resistance against ceftazidime but not meropenem.
Once mastered, the complete experimental protocol can be performed ina few weeks if done properly. After watching this video, you should have a good understanding of how to investigate the role of multicopy plasmids in the evolution of innovations in bacteria.
