Minispin f 45 12 11

To extract parasites from RBC, 250 µl blood was vortex mixed for 15 s with 250 µl 0.7% saponin in 140 mM phosphate-buffered saline pH 7.4 (PBS) and incubated 5 min at room temperature. After incubation 1500 µl PBS was added and samples were vortex mixed for 15 s and centrifuged for 2 min at 2579 g (6200 RPM, Minispin F-45-12-11, Eppendorf AG, Hamborg, Germany). After centrifugation, the supernatant was removed, and the lid and edges of the sample tube were carefully cleaned with a sterile cotton swap without touching the pellet. The pellet was then resuspended in 2000 µl PBS and samples were centrifuged for 1 min at 12,045 g (13.400 RPM, Minispin F-45-12-11, Eppendorf AG). The resulting pellet was spotted in a thin layer on clean steel target plates using a wooden toothpick and 1 µl matrix (alpha-cyano-4-hydroxycinnamic acid, Bruker Daltonics, Billerica, MA, USA) was applied to each sample spot. Samples were dried at room temperature before further processing. The sample preparation protocol was based on a method described by Baumeister et al. to separate intact Plasmodium parasites from erythrocytes [11 ].

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.