ABSTRACT
Many essential functions in biological systems, including cell cycle progression and circadian rhythm regulation, are governed by the periodic behaviors of specific molecules. These periodic behaviors arise from the precise arrangement of components in biomolecular networks that generate oscillatory output signals. The dynamic properties of individual components of these networks, such as maturation delays and degradation rates, often play a key role in determining the network's oscillatory behavior. In this study, we explored the post-translational modulation of network components as a means to generate genetic circuits with oscillatory behaviors and perturb the oscillation features. Specifically, we used the NanoDeg platform-A bifunctional molecule consisting of a target-specific nanobody and a degron tag-to control the degradation rates of the circuit's components and predicted the effect of NanoDeg-mediated post-translational depletion of a key circuit component on the behavior of a series of proto-oscillating network topologies. We modeled the behavior of two main classes of oscillators, namely relaxation oscillator topologies (the activator-repressor and the Goodwin oscillator) and ring oscillator topologies (repressilators). We identified two main mechanisms by which non-oscillating networks could be induced to oscillate through post-translational modulation of network components: an increase in the separation of timescales of network components and mitigation of the leaky expression of network components. These results are in agreement with previous findings describing the effect of timescale separation and mitigation of leaky expression on oscillatory behaviors. This work thus validates the use of tools to control protein degradation rates as a strategy to modulate existing oscillatory signals and construct oscillatory networks. In addition, this study provides the design rules to implement such an approach based on the control of protein degradation rates using the NanoDeg platform, which does not require genetic manipulation of the network components and can be adapted to virtually any cellular protein. This work also establishes a framework to explore the use of tools for post-translational perturbations of biomolecular networks and generates desired behaviors of the network output.
ABSTRACT
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.
