Additional file 1 of Optimal trade-off between boosted tolerance and growth fitness during adaptive evolution of yeast to ethanol shocks

Supplementary Material 1: Table S1. List of Saccharomyces cerevisiae strains. Table listing all S. cerevisiae strains used in this study, including parental strains, evolved clones, and genetically-modified yeasts. Table S2. Flow cytometry measurements in competitions with 24% v/v ethanol treatments. The proportion of each competitor is shown before (initial) and after the 24% ethanol treatment and outgrowth (final). Selection coefficients were calculated (S) and normalized with the data obtained for the progenitor. Two datasets were generated: one for the Progenitor, P1c, P2c, P3c without acclimation, and other with the same strains subjected to prior acclimation with ethanol shocks (see text). The data was used to generate the graphics of Fig. 1C and D. Table S3. Results from WGS and variant calling of P1c, P2c, P3c, and P4c. Mutations found in the evolved clones are shown. Coordinates are related to the PE-2_H4 parental strain (GenBank accession number GCA_905220315.1). Table S4. PCR oligonucleotides used in this study. Table S5. Flow cytometry measurements for competition assays 1–3. Data from competitions 1 (green), 2 (red), and 3 (blue) display the proportions of tester-GFP and the indicated competitor at the initial and final points of the assay. Selection coefficients (S) were calculated and normalized with the S obtained for the respective parental strain. For competitions 2 and 3 the S was expressed per cell doubling. Table S6. Microplate growth assay. OD600 was recorded every 15 min. on the Tecan Sunrise™ microplate reader. Each Strain was assayed in 3–4 replicates in YPS with 8% (v/v) ethanol, or in YPS without ethanol. Table S7. Fitness estimations for cyr1A1474T/usv1Δ. The PE-2_H4 (parental) and cyr1A1474T/usv1Δ were competed against the PE-2_H4-GFP through five passages in YPS with 8% (v/v) ethanol or without ethanol. Flow cytometry records of the competitors’ proportions and cell counting were taken at the initial and final points of each transfer, allowing the calculation of selection coefficients (S) during each passage and a cumulative S during serial transfers 2–5. By plotting the cumulative S per number of calculated generations at the end of each passage, selective coefficients per cell doublings (S/d) were calculated from the trendline fitted into the data points. Thereby an S per doubling was calculated for PE-2_H4 as -0.0079 for the competition with the PE-2_H4-GFP at 8% (v/v) ethanol and as 0.0049 for the competition in YPS without ethanol. Based on these numbers, a “S correction factor for PE-2_H4” was calculated accounting for cumulative cell doublings at each passage and used to normalize the cumulative S obtained for cyr1A1474T/usv1Δ in a competition with PE-2_H4_GFP. This normalization allows to express the cyr1A1474T/usv1Δ fitness (S) in comparison to PE-2_H4 and to discount any fitness effect of GFP expression in the PE-2_H4-GFP strain. The normalized cumulative S for cyr1A1474T/usv1Δ was plotted in Fig. 6C.