Figure 2. Fraction of WT and Δeps–ΔtasA strains changes during pellicle serial transfer.

In transfer method (a), the pellicle was harvested, disrupted (see Methods) and the suspension of spores and cells was directly used for inoculation of new medium. After harvest and disruption, the cells in transfer method (b) were heat-treated at 70 °C for 15 min to eliminate all vegetative cells. The resulting spore suspension was used to inoculate new medium and was incubated for 2–3 days to allow for pellicle formation. For transfer method (b), the gradual change of wild-type (grey) and Δeps–ΔtasA (white) ratio during the evolution experiment is also presented. The ratio was followed during evolution experiments using the disrupted pellicle suspension directly. In addition, the ratio was redetermined from frozen glycerol stocks for the second, eighth and tenth pellicles.