Figure 4.

Construction and characterization of a MuTMOS system with ribose as the master OFF signal. (A) A schematic of the MutMOS circuit that uses MalR-RbsR and native RbsR for detecting ribose. Engineered cells exposed to IPTG and ATc are switched to mCherry-ON state and GFP-ON state, respectively. An exposure to ribose turns off the expression of both mCherry and GFP. (B) Stochastic simulation of concentration changes of 4 repressors after induction (ON) by IPTG plus ATc (in shade). Ribose induces a subsequent OFF signal (in shade). A.U. stands for arbitrary units. The simulation was conducted by using the Gillespie algorithm. The levels of RbsR and MalR-RbsR increase while the levels of TetR and LacI decrease with the induction of IPTG plus ATc for the first 100,000 steps, and then maintained the equilibrium state even after the removal of ligands (upper row). The addition of ribose after 500,000 steps switched the toggle switches back to the state with high levels of LacI and TetR (lower row). (C) Experimental characterization of the MuTMOS system with ribose as the OFF signal. As shown on the first row, cells at the OFF state were exposed to IPTG only to switch to a stable mCherry-ON only state (left column); exposing cells only at the mCherry-ON state to ribose switched them back to a stable OFF state (right column). Similarly, results shown on the second row illustrates that ATc switched cells to a stable GFP-ON state (left), and ribose switched cells back to the OFF state (right). When cells were exposed to both ATc and IPTG (third row), cells became both mCherry-ON and GFP-ON (left), whereas ribose deactivated the expression of both mCherry and GFP to return cells back to a stable OFF state (right). Representative flow cytometry data are illustrated on the fourth row for the switching process between the state with both mCherry-ON and GFP-ON and the OFF state. All data points represent the mean ± S.D. of three biological replicates.