Research Paper Volume 8, Issue 10 pp 2568—2589

Figure 6. Quantifying the relative fitness of a long-lived mutant strain in a direct competition assay with a parental WT strain. His⁺ and His^- strains used in the direct fitness competition experiment are first cultured separately in the complete YP medium rich in amino acids, nucleotides and other nutrients until mid-exponential phase. Cells of the His⁺ strain are then mixed with the same number of cells of the His^- strain in liquid YP medium. After culturing the cell mixture for 7 days, an aliquot of cell suspension is diluted and plated on a solid YP medium. Following 2 days of incubation, colonies on each plate are replicated onto plates with the synthetic minimal YNB medium without amino acids and nucleotides. One of these plates contains leucine, lysine, uracil and histidine (it is called a His⁺ plate), whereas the other plate contains leucine, lysine and uracil (it is called a ″His^-″ plate). After 2 days of incubation at 30^oC, the number of CFU on ″His⁺″ and ″His^-″ plates is counted. The relative fitness of each His⁺ strain in a direct competition with the His^- is calculated as log₂ [(CFU^x _mutant/CFU^x _WT/(CFU⁰ _mutant/CFU⁰ _WT)], where: CFU^x is the colony count at the end of week x, whereas CFU⁰ is the colony count at initial inoculation of a mixed culture. The direct competition step of culturing a cell mixture for 7 days in liquid YP medium was repeated 6 times.