Titramax plateshaker

Evolution experiments were inoculated with ancestral colonies of IS+ and IS- strains grown in 3 ml of LB medium over night, after two washing steps in M9 medium without carbon source (M9 buffer) and a dilution of 1:200.
Bacterial cultures were grown in 200 µl liquid evolution medium with the indicated galactose concentrations in clear flat-bottom 96-well plates and shaken in a Titramax plateshaker at 750 rpm (Heidolph, Schwabach, Germany), allowing for a total population size of ~10⁸ colony forming units for the ancestral strain. Every day, populations were transferred to fresh plates using a VP408 pin replicator (V&P SCIENTIFIC, Inc, San Diego, CA) resulting in a dilution of ~1:820 (Steinrueck and Guet, 2017 (link)), corresponding to ~10 generations. Immediately after the transfer, growth and fluorescence measurements were performed in the overnight plates using a Biotek H1 plate reader (Biotek, Vinooski, Vermont). Thus, population phenotypes were measured every 10 generations.

Strains were pre-grown for 16 hr in M9CG medium without tetracycline and transferred to 96-well plates (200 µL/well). From there, we pin-diluted cultures with a VP408 pin replicator (V and P Scientific, San Diego, California, dilution factor ~1:820, tested with fluorescein) into fresh medium with different concentrations of tetracycline, incubated plates for 24 hr at 37°C on a Titramax plateshaker (Heidolph, Schwabach, Germany, 900 rpm), shook plates for 20 s at 1200 rpm and measured OD₆₀₀ with a H1 platereader (Biotek, Vinooski, Vermont). For obtaining fine-scale MIC measurements we tested tetracycline concentrations at intervals of 0.125 µg/mL. We defined MIC as the lowest drug concentration that yielded OD₆₀₀ ≤ 0.075 (plate reader units) in three replicates performed on different days.