Design of post-translational, ligand-controlled inverters and switches

Rational control over diverse, ligand-responsive output networks is a foundational challenge in synthetic biology, particularly for systems based on post-translational signaling. Here, we present the design and engineering of minimal, modular protein architectures for chemically responsive molecular inverters and digital switches. Our inverter design modifies the plant-derived PYR1-HAB1 chemically inducible dimerization module by incorporating a constitutive activator, which is then competitively displaced by the ligand-bound PYR1 receptor, converting the native ‘dimerization-on’ mechanism into a ‘signal-off’ inverter. We establish key design features and demonstrate predictable tuning of the inverter’s transfer function, including its maximum output, half-maximal inhibitory concentration, and minimum output, solely by adjusting protein stoichiometry. The architecture is modular, enabling plug-and-play response to diverse, user-defined drug-like small molecules. We show that an inverter biosensor for an environmental contaminant functions in engineered living cells with a low nanomolar sensitivity. Additionally, we convert the PYR1-HAB1 sensor into a digital switch by adding an engineered molecular titrant to the system. Overall, this work provides a generalizable, minimal, and tunable protein scaffold for programming complex, post-translational signaling logic, significantly expanding the toolkit for sophisticated biological circuit design.