Gu, LiangcaiPan, Yuxin2025-01-232025-01-232024Pan_washington_0250E_27700.pdfhttps://hdl.handle.net/1773/52725Thesis (Ph.D.)--University of Washington, 2024Methods for de novo engineering of protein binders into chemically induced dimerization (CID) systems offer new opportunities for developing genetically encoded sensors for drugs and metabolites that lack suitable natural binders. However, integrating these binders into genetically encoded fluorescent sensors (GEFSs) remains largely unexplored. Here, we present a pipeline that efficiently selects synthetic CIDs and integrates them with a fluorescent domain to create single-protein GEFSs suitable for solution-based assays and mammalian cell applications.As proof of concept, we used this pipeline to generate synthetic CID pairs from monobody and nanobody scaffolds and incorporated them into circularly permuted green fluorescent protein-based GEFSs. To optimize sensor performance, we created a library of 361 unique linker variants and conducted lysate-based screening to identify the most effective configurations. The resulting sensors exhibited a ΔF/F₀ exceeding 100%. Furthermore, these sensors retained functionality in HEK293T cells when localized to the plasma membrane, cytoplasm, or ER lumen. We also adapted this approach to additional optical domains, such as circularly permuted HaloTag , demonstrating its broad applicability. Our results establish a foundation for expanding synthetic GEFSs, significantly enhancing the small molecule and peptide GEFS toolkit and reducing reliance on natural proteins and derivatives.application/pdfen-USnoneGenetically encoded biosensorPhage biopanningProtein engineeringBiochemistryBiological chemistryThesis