Fig. 3: Enhancing drug-inducible transcriptional control.

a Circuit design for rtTA-mediated activation of TRE3G. b Dox-dependent activation of a TRE3G-regulated LUC reporter (N = 3 biological replicates). c Titers of VLVs propagated on cells expressing VSVG under the control of TRE3G at 100 ng/mL dox (gray box) (N = 4 biological replicates). Red bars indicate the RT-qPCR NTC signal. Statistical comparisons were made using two-tailed unpaired t-tests. d Allele frequency of the major variants identified during long-term propagation on minimal concentrations of dox. e Dox-sensitivity of evolved rtTA variants (N = 3 biological replicates). f Activation of evolved rtTA variants in embryoid bodies at DIV6; scale bar, 200 μm. g Quantification of mCherry fluorescence in embryoid bodies presented in (f) (for D5N/M59I embryoid bodies, N = 1 biological replicate for 0 ng/mL dox, N = 3 biological replicates for 125 and 1000 ng/mL dox; N = 4 biological replicates for all WT embryoid bodies). Error bars represent mean ± SEM. Statistical comparisons were made using a two-way ANOVA with Šídák’s multiple comparisons test to generate adjusted P values. h Circuit design for rtTA-mediated inhibition of mCherry expression. i Dox-sensitivity of evolved rtTA variants in E. coli (N = 2-3 biological replicates). Schematics for panels (a) and (h) were created with BioRender.com. Source data are provided as a Source Data file.