This method can help answer key questions in the microbiology field about how often plasmid DNA can be spread among different bacteria. The main advantage of this technique is that by reducing the background, we can detect small differences in the conjugation frequency with a high resolution. Demonstrating the procedure will be Kosuke Yanagiya, a graduate student from my laboratory.
To calculate the conjugation frequency by most probable number, filter mate one milliliter from an overnight donor culture with one milliliter from an overnight recipient culture for 45 minutes at 30 degrees Celsius, as just demonstrated. While the co-culture is incubating, serially dilute the original donor and recipient cultures for plating in triplicate onto donor Luria broth, or LB, plus kanamycin, or recipient LB plus gentamycin plates for a two-day culture at 30 degrees Celsius. At the end of the incubation, resuspend the culture on the filter in sterile LB containing kanamycin and gentamycin for serial dilution in a 96-well cell culture plate in quadruplicate.
After two days at 30 degrees Celsius, manually count the colony-forming units, or CFUs, on the donor and recipient agar plates and the number of wells in which the transconjugants grow by light microscopy. To calculate the most probable number and its deviation, open the MPN calculation program. Enter the name and date of the experiment, the number of the test series, and the maximum number of dilutions in row seven of the program sheet of the spreadsheet file.
In the automatically-produced input data tables, enter the formula in the dilution factor D column, 0.01 in the volume in milliliters or GW column, and four in the number of tubes N column. Enter the number of wells in which the transconjugants grew at each sample dilution and click calculate results to obtain the results as most probable number per milliliter, and the upper and lower 95%confidence limits. Then divide the number of transconjugants by the number of donor and recipient colony forming units per milliliter to calculate the conjugation frequency of the plasmids.
In this representative experiment, the conjugation frequency of both plasmids increased at higher stirring rates with a maximum difference in conjugation frequency observed between zero and 400 RPM for cultures introduced to the pBP136:gfp plasmid, and between zero and 200 RPM for cultures that were introduced to the pCAR1:gfp plasmid. To determine the density of recipient cells required to compare the probability of conjugation, mating assays were performed with different donor and recipient densities. In this representative experiment, pBP136:gfp transconjugants were detected in 100%of the wells containing one times 10 to the third CFU of donor and one times 10 to the fifth to one times 10 to the seventh CFU of recipient, and those with one times 10 to the second CFU of donor and one times 10 to the sixth to 10 to the seventh CFU of recipient, indicating that the cell density was too high.
Mating assays with 10 CFU of donor and 10 to the sixth or 10 to the fifth CFU of recipient, however, resulted in a decreased number of transconjugant positive wells. Thus, 10 to the fifth CFU of recipient was predicted to be required for mating with a single donor cell. Mating assays with pCAR1:gfp at different densities of donor and recipient strains demonstrated similar findings, although overall the percentages of transconjugant positive wells were much lower than those of pBP136:gfp.
Assuming that the donor and recipient cells can attach to each other similarly, the probability of conjugation initiation for the pCAR1 donor was lower than that for the pBP136 donor. Based on these results, it was estimated that 10 to the seventh CFU of recipient was required for a single donor cell sorted by FACS. After counting the numbers of transconjugant positive wells, the percentage of transconjugant positive wells for pBP136:gfp was determined to be larger than that for pCAR1:gfp, demonstrating a more than 36-fold difference in the probability of donor initiated conjugation between these two plasmids.
While attempting the procedure, it's important to remember to always dilute the bacterial mixtures carefully.
