Engineering mammalian cells holds great promise for a variety of biomedical applications, ranging from the design of model systems to study complex biological processes underlying human diseases, to the development of cell-based therapies and the production of therapeutic biomolecules. Cell engineering requires sophisticated molecular tools that can interface with cellular systems, but also provide orthogonal functionalities, to achieve precise control over complex gene networks. In an attempt to expand the mammalian synthetic biology toolbox for applications in the study of protein misfolding diseases, my research seeks to design and construct orthogonal gene regulators and genetic circuits to monitor protein folding and degradation. To develop a cell-based sensor for monitoring protein aggregation, I engineered a split transcriptional repressor that links protein aggregation to expression of an easily detectable reporter. I designed a set of two-fragment tetracycline repressor (TetR) variants that can function as transcriptional AND gates in both bacteria and mammalian cells. I built a protein solubility sensor by co-expressing the large “detector” fragment of the split TetR and a small “sensor” fragment fused to the target protein, and demonstrated that protein aggregation can be detected by monitoring complementation between the “detector” and “sensor” fragments. This split TetR represents a novel genetic component that can be used in bacterial as well as mammalian synthetic biology and a much-needed cell-based sensor for monitoring protein conformation in complex cellular environments. To develop a tunable, hysteretic sensor for detection of proteasomal degradation, I built a genetic circuit (Hys-Deg) based on a self-activation loop consisting of a tetracycline-controlled transactivator (tTA) variant engineered to interface with the ubiquitin proteasome system (UPS). Guided by predictive modeling, I demonstrated that control of the hysteretic response is achieved by modulating the ratio of expression of constitutive to inducible tTA that generates the self-activation loop. I also showed that the system can be finely tuned through dosage of tetracycline to calibrate the circuit for detection of desired levels of UPS activation. This study establishes the design rules for building a hysteretic circuit with an autoregulatory feedback loop and provides a synthetic memory module that can be integrated into regulatory gene networks to study and engineer complex cellular behaviors.