Analysis and characterization of the IncFV plasmid pED208 transfer region
E. coli XK1200 and ED24 were used as donor and recipient strains, respectively, in all the mating experiments. Mating experiments using donor cells containing R100 or R100-1, encoding spectinomycin resistance, used ED24 carrying the non-mobilizable plasmid pT7-5 (ApR) as the recipient strain. Donor and recipient cultures were grown to mid- and late-exponential phase, respectively, in LB with appropriate antibiotics. Fifty μl of donor and 200 μl of recipient cells were mixed in 1ml of 37 °C pre-warmed LB medium and incubated at 37 °C for 30 min. Mating was terminated by vortexing vigorously and putting the cells immediately on ice to prevent further conjugation. After serial dilutions in cold SSC (0.15 M sodium chloride, 0.015 M sodium citrate, pH 7.0), 10 μl portions of each dilution were spot-dropped onto selective plates containing combinations of antibiotics to select for donors and transconjugants. Plates were dried and incubated at 37 °C overnight. Plasmid transfer efficiency was calculated as the number of transconjugant colonies divided by the number of donor colonies. All mating assays were repeated 2–3 times with the results being within one log unit of each other.
Jun Lu, Jan Manchak, William Klimke, Colin Davidson, Neville Firth, Ronald A. Skurray and Laura S. Frost
Characterization of the Opposing Roles of H-NS and TraJ in Transcriptional Regulation of the F-Plasmid tra Operon
Mating assays. MC4100 and PD32 donor cultures containing pOX38-Tc, Flac, or Flac traJ90, along with ED24 recipient cell cultures, were diluted 200-fold from standing overnight cultures grown at 37°C into fresh LB broth and grown to an OD600 of approximately 0.6 at 37°C with shaking. Both donor and recipient cells of OD600s of 0.1 were mixed in fresh LB broth in a final reaction volume of 1.0 ml. The reaction volumes were incubated at 37°C for 1 hour, vortexed vigorously to disrupt mating pairs, and then placed on ice for 5 minutes to halt all mating. The reaction volumes were then serially diluted in SSC buffer (150 mM NaCl, 15 mM Na-citrate) and plated on the appropriate media to select for donors or transconjugants. Donors were selected by growth on LB agar containing streptomycin. Transconjugants containing pOX38-Tc were selected for on LB agar plates containing tetracycline and spectinomycin. Transconjugants containing Flac or Flac traJ90 were selected by growth on L1 synthetic medium (1x M9, 0.4% lactose, 5 mM MgSO4, 1.5% agar) containing spectinomycin. Plates were incubated for 1 to 2 days at 37°C and scored for growth. Mating efficiency was expressed as the number of transconjugants per donor cell.
William R. Will and Laura S. Frost