Plasmid Fitness Assay in E. coli

We conducted pair-wise competitions to assess the relative fitness of the plasmid bearers compared to GFP-labeled plasmid-free MG1655 (60 (link)). Overnight cultures were created from LB agar plates in 10 mL LB (plasmid-free) and 5 LB with 4 µg/mL cefotaxime (transconjugants) from single colonies of the plasmid-free strain and the biological triplicates of the transconjugants. All cultures were diluted to approximately 1 × 10⁹ cells/mL in PBS. Transconjugant cultures were then centrifuged for 5 minutes at 10,000 rpm (Eppendorf MiniSpin F-45–12-11), resuspended in 1 mL PBS, centrifuged for 5 minutes at 10,000 rpm, and resuspended in 1 mL LB. Plasmid-free cultures were then centrifuged for 10 minutes at 3,600 rpm (Thermo Scientific Megafuge 40R TX-1000), resuspended in 28 mL PBS, centrifuged for 10 minutes at 3,600 rpm, and resuspended in 28 mL LB. All cultures were then serially diluted to approximately 1 × 10⁵ cells/mL before 50 µL plasmid-free and 50 µL transconjugant were mixed in 5 mL LB and incubated for 24 hours at 37°C. Samples of 0-hour and 24-hour mixed populations were stored at −80°C until flow cytometry analysis.