Ultrasensitive Scaffold-Dependent Protease Sensors with Large Dynamic Range

The rational construction of synthetic protein switches with predefined input output parameters constitutes a key goal of synthetic biology with many potential applications ranging from metabolic engineering to diagnostics. Yet, generally applicable strategies to construct tailor engineered protein switches have so far remained elusive. Here, we use SpyTag/SpyCatcher-mediated protein ligation to engineer modularly organized, scaffold-dependent protease sensors that exploit a combination of affinity targeting and protease-inducible protein protein interactions. We use this architecture to create a suite of integrated signal sensing and amplification circuits that can detect the activity of alpha-thrombin and prostate specific antigen with a dynamic range covering 5 orders of magnitude. We determine the key design features critical for signal transmission between protease-based sensors, transducers, and actuators.