BerkeleyConjugation

From 2006.igem.org

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.

Analysis and characterization of the IncFV plasmid pED208 transfer region

Jun Lu, Jan Manchak, William Klimke, Colin Davidson, Neville Firth, Ronald A. Skurray and Laura S. Frost