Figure 3. Protease-driven control of a synthetic toggle switch.

(a) Schematic of the synthetic toggle switch in which reciprocal transcriptional repression by TetR and LacI form a bistable circuit. GFP and mCherry serve as fluorescent reporters for the LacI+ and TetR+ toggle states, respectively. Addition of a pdt tag to LacI enables a protease-driven switch from the GFP+ to the mCherry+ state. (b) Flow cytometry scatter plots show GFP and mCherry fluorescence 0, 4 and 8 hours after mf-Lon expression from the inducible promoter P_BAD. Degradation of LacI-pdt#3 causes the toggle to switch from the GFP+ state to the mCherry+ state by 8 hours, while the untagged toggle remains in the GFP+ state. Magenta lines indicate the gate parameters used to define the GFP+ and mCherry+ states: cells bounded in the lower left quadrant are considered negative for both GFP and mCherry. (c) The percentage of cells in the mCherry+ state following mf-Lon induction with 1 mM arabinose. Data collected by flow cytometry were measured using the parameters shown in (b) and represent the mean of three biological replicates. For all LacI-pdt variants, P < 0.001 when compared to untagged LacI at 24 hours after induction. Error bars show the standard deviation. See Supplementary Figure 6 for data showing that non-induced strains did not shift to mCherry+.