Figure 2. Ligand-dependent transcriptional activation.

(a) TF-biosensor construction from a conditionally destabilized LBD, a DNA-binding domain and a transcriptional activation (TAD) domain. (b) Positions of conditionally destabilizing mutations of Gal4 mapped to a computational model of Gal4-DIG₀ homodimer. Residues are shown as colored spheres and key interactions are highlighted in insets. The TAD is not shown. (c) Concentration dependence of response to digoxin for digoxin TF-biosensors driving yEGFP expression. (d) Concentration dependence of response to progesterone for progesterone TF-biosensors driving yEGFP expression. (e) Time dependence of response to 250 μM digoxin for digoxin TF-biosensors. (f) Time dependence of response to 50 μM progesterone for progesterone TF-biosensors. (g) Time-dependent response to withdrawal of 250 μM digoxin for digoxin TF-biosensors. (h) Time-dependent response to withdrawal of 50 μM progesterone for progesterone TF-biosensors. In c-f, (-) indicates cells lacking biosensor plasmids and error bars represent s.e.m. of three biological replicates. Marker symbols in e and g are the same as in c. Marker symbols in f and h are the same as in d.

DOI: http://dx.doi.org/10.7554/eLife.10606.006

Figure 2—figure supplement 1. Improvements to TF-biosensor response.

Digoxin-dependent expression of yEGFP by G-DIG₁-V TF-biosensors either (a) containing VP64 or VP16 as the TAD and expressed from a CYC1 promoter or (b) containing a VP16 TAD and expressed from a CYC1, ADH1, or TEF1 promoter. (c) Individual mutations identified in a FACS analysis of an error-prone PCR library of G-DIG-V biosensors were tested for their effect on biosensor function using digoxigenin. Transformants were analyzed in an yEGFP yeast reporter strain containing a deletion of PDR5 (PyE14). Improvements in fold activation relative to parental sequences were localized to mutations in Gal4. (d) R60S and L77F mutations found in Gal4 were introduced into G-DIG₁-V, G-DIG₂-V, and G-PRO₁-V. In each case, the Gal4 mutations had the effect of lowering the amount of luciferase activity in the absence of the relevant ligand. In a-d, error bars represent s.e.m. of three biological replicates.

DOI: http://dx.doi.org/10.7554/eLife.10606.007

Figure 2—figure supplement 2. Population responses to cognate ligand for cells bearing TF-biosensors.

(a) yEGFP fluorescence of DIG₀-GFP upon addition of digoxin or DMSO vehicle (left) and yEGFP fluorescence of DIG₁-GFP upon addition of digoxin or DMSO vehicle (right). (b) yEGFP fluorescence of PRO₀-GFP upon addition of progesterone or DMSO vehicle (left) and yEGFP fluorescence of PRO₁-GFP upon addition of progesterone or DMSO vehicle (right). Data for each condition are presented for a single representative biological replicate with 20,000 events measured by flow cytometry.

DOI: http://dx.doi.org/10.7554/eLife.10606.008