Fig. 1: On and off dynamics of inducible transcriptional systems.

A The reporter for transcriptional activity consists of an inducible promoter and the fast-folding yellow fluorescent protein yEVenus gene fused to a constitutive degron (PEST) and the ADH1 terminator. B–N Time courses of activation and deactivation for different inducible systems sorted in descending order by peak average strength. Induction starts at t = 0 h and finishes at t = 3.5 h. The blue background represents the induction period. Expression is quantified in maxGAL1 units, where 1 maxGAL1 corresponds to the steady-state expression of GAL1pr. Black lines show the average of the mean cellular expression and standard deviation (for standard error of the mean, see Supplementary Fig. S3). Colored lines show different representative single-cell time courses. For the light-inducible systems, fluorescence was not measured prior to induction in order to avoid possible activation by the light source used for fluorescent protein excitation. El222 refers to the WT-El222 transcription factor inducing LIP under 20% or 80% light intensity, as indicated. strongLOV refers to the Glu84Asp El222 mutant introduced in this article inducing LIP under 20% light. GLIP is induced by El222 under 80% light. Due to the high sensitivity of strongLOV to the excitation light used for the yEVenus fluorescence measurements, quantification of the off dynamics by microscopy was done using ymScarletI as a reporter (see Fig. 7). Dashed black lines in (C, D, J) represent averages of the mean cellular expression measured using LIP-ymScarletI as a reporter. Induction of the PhyB-PIF3 system was performed in synthetic complete media supplemented with raffinose as a carbon source, to avoid inhibition by the glucose-induced GAL1pr repressor Mig1. The numbers of analyzed cells for each plot are given in Supplementary Table 14.