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Mammalian cells process information through coordinated spatiotemporal regulation of proteins. Engineering cellular networks thus relies on efficient tools for regulating protein levels in specific subcellular compartments. To address the need to manipulate the extent and dynamics of protein localization, we developed a platform technology for the target-specific control of protein destination. This platform is based on bifunctional molecules comprising a target-specific nanobody and universal sequences determining target subcellular localization or degradation rate. We demonstrate that nanobody-mediated localization depends on the expression level of the target and the nanobody, and the extent of target subcellular localization can be regulated by combining multiple target-specific nanobodies with distinct localization or degradation sequences. We also show that this platform for nanobody-mediated target localization and degradation can be regulated transcriptionally and integrated within orthogonal genetic circuits to achieve the desired temporal control over spatial regulation of target proteins. The platform reported in this study provides an innovative tool to control protein subcellular localization, which will be useful to investigate protein function and regulate large synthetic gene circuits., (© The Author(s) 2021. Published by Oxford University Press.)

Mammalian cells rely on complex and highly dynamic networks that respond to environmental stimuli and intracellular signals and maintain homeostasis. The use of synthetic orthogonal circuits for detection of dynamic behaviors has been limited by the remarkable stability of conventional reporters. While providing an appealing feature for signal amplification, the long half-life of reporters such as GFP is typically not ideal to measure transient signals and dynamic behaviors. This chapter explores the use of post-translational regulation for the design of input-dependent circuits that produce output signals with enhanced dynamic range and superior dynamic resolution of the input. Specifically, we report the use of the NanoDeg-a bifunctional system that mediates proteasomal degradation of a cellular target with high specificity and control over rate of decay-to achieve input-dependent depletion of a GFP reporter. Feedforward loop topologies were explored and compared to conventional reporters placed directly under control of the input to identify the ideal circuit architecture that allows placing both the GFP output and the GFP-specific NanoDeg under control of a common input and regulate GFP levels not only through input-dependent transcriptional activation but also input-dependent degradation. The circuit design was implemented experimentally by building a heat-sensitive reporter and exploring the design features that result in detection of the cell response with maximal output dynamic range and dynamic resolution of the heat shock. The method reported provides the design rules of a novel synthetic biology module that will be generally useful to build complex genetic networks for enhanced detection of highly dynamic behaviors., (© 2019 Elsevier Inc. All rights reserved.)