Input-dependent post-translational control of the reporter output enhances dynamic resolution of mammalian signaling systems

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.