Three ml of both early-log-phase cultures of donor pK18C1C2-bearing E. coli S17-1 and recipient P. putida KT2440 grown in LB medium were mixed and centrifuged to harvest cells; the pellet was resuspended in 100 μl of LB medium, which was then dropped on a filter placed on an LB agar plate that was then incubated at 37 ฐC overnight to allow conjugation to occur.