PROJECT SUMMARY/ABSTRACT Even with recent advances in synthetic biology, it remains a major challenge in developing genetic circuits that involve multiple inputs and outputs. This is because natural genetic systems are only capable of connecting one single chemical input to one specific promoter to control gene expression. This poses a significant barrier in creating engineered organisms with complex signal response behavior for biomedical applications. The long-term goals of this research team are to establish robust strategies for constructing biological parts of genetic circuits, and to use these parts to expand researchers’ ability in engineering new cellular functions for biomedical applications. In their recent progress, the team established a module swapping strategy for building genetic sensors from regulators in the LacI and TetR families and they harnessed these engineered sensors to develop several novel genetic circuits. The two directions in this proposed research represent important steps toward the team’s long-term goals in the next five years. The first direction is to advance the capabilities in engineering transcriptional regulators as modular biosensors. Specifically, the team plans to 1) establish design principles of modifying regulators for enhancing their performance as biosensors and 2) apply module swapping to a wide range of regulator families. The central hypothesis is that each regulator within a family contains a ligand-binding module (LBM) and a DNA-binding module (DBM) for the purpose of detecting an input signal and for interacting with a promoter, respectively; if key module-module interactions are maintained, LBMs and DBMs from different regulators can be mixed and matched to create hybrid regulators with new combinations of input sensing and DNA recognition properties. For their second direction, the team proposes to harness hybrid regulators to explore novel circuit designs in various organisms, aiming to meet emerging needs in biomedical fields. This effort includes developing cellular devices to continuously and simultaneously monitor a range of toxic pollutants, which provides a means to assess the intake of toxicants that are commonly found in contaminated food and water. As an Early Stage Investigator, the PI and his team have already generated significant progress on both proposed directions, showing that they are highly qualified to pursue the proposed projects. The contribution of this project is expected to be the establishment of design principles for creating modular parts from regulators in many families and the advancement in genetic circuit design and implementation. This contribution will be significant because it is expected to release many new possibilities in circuit topologies for biomedical uses, including monitoring devices that will be created in this program. The overall approach is innovative because it represents a new way of using protein engineering and cellular engineering approache...