Browsing by Author "Kuypers, Brianna"

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    Nanobody-mediated dynamic and spatial control of cellular proteins for mammalian gene circuit engineering
    (2019-11-22) Kuypers, Brianna; Segatori, Laura
    Cellular information processing in mammalian cells relies on sophisticated and highly dynamic mechanisms. Unravelling these mechanisms remains a challenging endeavor and has limited de novo design of biological functions. Controlling cellular phenotypes requires precise control of protein concentration and localization, which is typically realized through the design of genetic networks orthogonal to the cellular circuitry. This work describes my efforts to develop tools for achieving temporal and spatial control over cellular proteins through post-translational regulation that can be generally used to characterize and manipulate the complex genetic networks that regulate mammalian cells. I first illustrated the use of a nanobody-based platform designed to control degradation of target proteins (the NanoDeg) to improve the dynamic range and temporal resolution of input-dependent reporter systems. I then explored integration of a reporter-specific NanoDeg into a genetic circuit through a feedforward loop to generate an improved heat-shock reporter system. To enhance temporal control of genetic networks, I investigated strategies to use the NanoDeg for controlling the oscillatory behavior of a range of different circuit topologies. Finally, I developed a nanobody-based platform for controlling the subcellular localization of target proteins. Using this strategy, I experimentally demonstrated selective target localization to multiple cellular compartments and investigated different circuit architectures for dynamic control of target localization to different compartments. Combining nanobody-mediated localization and degradation with orthogonal transcriptional regulation I developed a nanobody-based toolkit to achieve spatial and temporal control of target protein levels.