Fig. 3: Directed evolution of the S/N ratios of Tet-ON switches.

a Random mutations were introduced into the rTetTA-encoding expression cassette of the Tet-ON switch, and the resultant library was subjected to parallel operation of OFF/ON selections under different selection conditions. The resultant cell populations were evaluated for Dox-dependent fluorescence shifts to screen for the conditions under which switch variants were enriched. The green and blue areas indicate the relative abundance (%) of (respectively) the unwanted behaviour, i.e., leaky variants (cells with GFP fluorescence more than 10⁴ in the absence of Dox) and the inactive variants (cells with GFP fluorescence >10⁴ in the presence 10 µg/mL Dox). Relative abundance (%) of the switching variants was obtained by subtracting both of these two numbers from 100%. Dashed lines indicate the histogram obtained from yeast strains carrying plasmid without rTetTA. b A 96-well-plate-based fluorescence screening of the Tet-ON variants. c Transfer function of the selected mutant. Error bars represent the mean ± SD of three independent experiments. The 50% effective concentration (EC₅₀) values were calculated from the dose–response curve fitted to the Hill equation by the least-squares method. The top panel represents the fluorescence image for the cell pellets of yeast strains harbouring wild-type and evolved Tet-ON switch (rTetTA_{K8N, L13L}) incubated with or without 10 µg/mL Dox. Source data are available in the Source Data file.