A reversible genetic NOR gate in plants using translational repression

Abstract

A proof-of-concept reversible genetic logic circuit in tobacco plants is presented. The genetic circuit implements a Boolean NOR function using a Cas6-based translational repression system, with exposure to estradiol and ethanol as inputs, and expression of GFP as the output. Expressed in the presence of the inducers, two Cas6 proteins are used to selectively prevent the translation of GFP. The circuit yields a 40-90% reduction in GFP expression in the presence of the inducers. A mathematical model of the circuit's mechanism of action is proposed and validated using experimentally acquired data. The employed genetic circuit design methodology is versatile, simplistic, and analogous to PMOS-based pass-transistor logic used in electronic circuit design, making it possible to design complex logic circuits without extensive biological expertise. Lowering the barrier to entry, this methodology can help improve the use of synthetic biology and its integration with other systems.

Keywords: Boolean logic; Cas6; functional sensing; genetic circuits; synthetic biology; translational repression.