Plasmid Evolution and Stability Experiments

Five parallel lineages of each plasmid-carrying strain were passaged once daily, in selective medium to ensure plasmid inheritance during the evolution experiment, and subsequently without selection to assess plasmid stability. Serial passaging was performed in 96-well plates containing 150 µl LB medium. Each day of the experiment, 1 µl culture was transferred to the corresponding well of a new plate by pin replication. Plates were incubated at 37°C and subjected to medium shaking at 400 rpm. The plasmid selection (cefuroxime, 16 µg/ml) used for the evolution experiment did not reduce the growth rate of plasmid bearing strains compared to growth in LB without antibiotics.