Moscow high school evolution protocol

Evolution experiments were carried out via batch transfer. Every seven days, yeast were transferred on a sterile cotton swab from a saturated culture to a tube of fresh media containing clotrimazole. Clotrimazole was obtained from an over-the-counter 1% solution dissolved in 70% isopropanol. Cultures were maintained in 5 mL of YPD medium with G418 (200 mg/L) and clotrimazole at room temperature without aeration. Student groups maintained one replicate of an assigned strain throughout their Honors Biology course (grade 10; first biology course). Yeasts were initially exposed to a much higher dose of clotrimazole (10 µM), which prevented visible growth of the ancestor over short time scales (Fig. 1b), but permitted growth over the extended time between transfers. The dose of clotrimazole was increased at student discretion at 2× intervals (e.g. 20, 40, 80 µM, etc.). Cultures were maintained for up to 12 weeks.

Overview of evolution experiment and sequencing. a) Outline of experiment. Yeasts were propagated in increasing concentrations of clotrimazole for several weeks. Clones from these experiments were sequenced to identify mutations that occurred during the experiment. b) Maximum measured clotrimazole tolerance of clones isolated from experiments at different schools and timepoints. c) Number of point mutations in sequenced clones.