Engineering a genetic toggle switch in plants

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Mauricio Antunes, Alberto J. Donayre, June I. Medford

COLORADO STATE UNIVERSITY, United States

We have previously engineered a synthetic genetic circuit that enables plants to serve as customizable biological input-output systems. This circuit uses computationally-designed receptors that bind a ligand of interest (input), initiate a signal through a synthetic signal transduction module, producing a readout (output). In our current design, production of a visible readout requires continuous exposure of plants to the ligand. Ideally, this system should be capable of reporting short, transient exposures to a ligand, essentially “remembering” the exposure event. This memory function can be introduced with the addition of a toggle switch circuit, consisting of two mutually repressible promoters that control expression of their respective transcriptional repressors. By controlling the levels of these repressors, this circuit can exist in one of two stable states. This feature not only adds memory to any synthetic circuit, but it also allows input-output systems to be reset. Therefore, we engineered and implemented a plant-functional genetic toggle switch that can be toggled between two stable output states (ON vs. OFF); toggling is controlled by two estrogen inducers. We started by developing novel transcriptional repressor-promoter pairs that function in plants. We then quantitatively characterized the behavior of these pairs by transient expression in isolated plant cells (protoplasts), and used the data obtained in vivo to build a computational model of the plant genetic toggle switch. Based on this model, we generated transgenic plants containing a genetic toggle switch circuit that uses two of these repressor-promoter pairs in a mutually repressing architecture. The switch behavior of this circuit was followed by monitoring in vivo production of a luciferase reporter gene (output) in response to the inducers. By modifying the output of this toggle switch, we are using this circuit to control plant traits with bioenergy applications. Funding: ARPA-E, US-DOE