Figure 4. Dynamic measurements of the induction of two osmostress responsive promoters in the same cells.

(a) Microscopy images of cells carrying pGPD1-dPSTR^R (RFP channel) and pSTL1-dPSTR^Y (YFP channel) stimulated with 0.2 M NaCl. The two reporters are built with two orthogonal pairs of SynZips. The arrowheads indicate the nuclei with accumulated FPs, highlighting the different timings of accumulation of each dPSTR. Scale bar, 5 μm. (b) Quantification of the nuclear enrichment of pGPD1-dPSTR^R (green, left axis) and pSTL1-dPSTR^Y (purple, right axis) in course of time (N_C=260). (c) Histograms of the time needed to overcome the expression threshold for cells expressing the indicated promoter. (d) Correlation between the expression output of pGPD1-dPSTR^R and the one of pSTL1-dPSTR^Y in single cells (R²=0.48). The dashed lines represent the expression thresholds for each dPSTR. The inset is showing the fraction of the population expressing either pGPD1 alone (cyan), pSTL1 alone (red), both promoters (blue) or none (orange). (e) The delay between pGPD1 and pSTL1 expression in cells that express both dPSTRs calculated from the difference in time to overcome the expression threshold for both reporters. Positive times represent cells where pGPD1 overcomes the expression threshold first (green area, 76% of the cells expressing both promoters), and negative or null times indicates that pSTL1 is expressed before or at the same time as pGPD1 (purple, 24%).