Research Paper Volume 8, Issue 10 pp 2568—2589

Figure 7. Validation of the developed assay for quantifying the relative fitness of a long-lived mutant strain in direct competition with a parental WT strain. The WT strains BY4742 (His^-) and BY4739 (His⁺, but otherwise isogenic to BY4742) were cultured separately in the complete YP medium containing 0.2% glucose, 2% glucose or 1% ethanol glucose until mid-exponential phase. Another pair of strains whose relative fitness was measured, namely the long-lived mutant strain dbp3Δ (His^-; is isogenic to BY4742) and the WT strain BY4739 (His⁺), was also cultured separately in YP medium containing 0.2% glucose 2% glucose or 1% ethanol glucose until mid-exponential phase. Cells of the His⁺ strain were mixed with the same number of cells of the His^- strain and then co-cultured for 7 days in liquid YP medium initially containing different carbon sources. Cells of the His^- and His⁺ strains pre-cultured separately on 0.2% glucose were subjected to direct fitness competition by being cultured together on 0.2% glucose (A) or 1% ethanol (C). Cells of the His^- and His⁺ strains pre-cultured separately on 2% glucose were subjected to direct fitness competition by being cultured together on 2% glucose (B) or 1% ethanol (D). Cells of the His^- and His⁺ strains pre-cultured separately on 1% ethanol were subjected to direct fitness competition by being cultured together on 1% ethanol (E). After culturing the cell mixture for 7 days, an aliquot of cell suspension was used to measure the relative fitness of the His⁺ strain in direct competition with the His^- strain (as described in ″Materials and Methods″). The direct fitness competition step of culturing a cell mixture for 7 days in liquid YP medium was repeated 6 times.