(725a) A Hysteretic Mammalian Genetic Circuit for Detection of Proteasomal Degradation

The design and construction of gene networks that exhibit complex dynamics remains a major challenge in mammalian synthetic biology. Hysteretic bistable networks require a reduction of stimulus greater than the amount that is required to trigger a system to shift from one state to another, in order to reverse the shift, giving rise to the ability to imprint a memory of gene expression history after induction decreases. Here we report the development of a hysteretic cell-based platform to detect protein degradation. Specifically, we developed a hysteretic genetic circuit that interfaces with the ubiquitin proteasome system (UPS), which is a complex cellular machinery that is dedicated to the degradation of misfolded or otherwise damaged proteins through the proteasome, and links UPS activity to an easily detectable output signal and presents cellular memory of proteasomal degradation. This genetic circuit is comprised of a genetic inverter and a self-activation loop that enables self-amplification of an engineered transcriptional regulator (tTA). The engineered tTA is rationally designed to function as a substrate of proteasomal degradation, thus translating the changes in UPS activity to the expression of a reporter protein. Predictive modeling was used to investigate the design rules of this expression system and build genetic circuit that exhibits bistability, thus presenting memory of modulation of UPS activity. This property enables sustaining the output signal over time, thereby facilitating detection and discovery of UPS modulators. The hysteretic behavior of this circuit was confirmed by generating stable cell lines expressing the relevant parts of the circuit and demonstrated in response to tetracycline dosage. We anticipate that this system will respond to modulation of UPS activity in cell culture arising from proteasome activators and inhibitors. By providing a cell-based platform for sensitive detection of UPS activity, this hysteretic genetic circuit provides a powerful tool to discover UPS modulators for a wide range of biomedical applications.