Modularity is an important property of engineered systems, yet it is debatable whether it is a general property of natural bio-molecular systems. Discovering the extent of modularity and understanding its mechanisms is one of the most important open research problems in systems biology. Furthermore, the long term success of synthetic biology critically depends on the ability to implement modular systems in such a way that the properties of individual components do not change unpredictably upon their interconnection. Our proposed research seeks to understand the mechanisms of modularity in regulatory networks using a combined theoretical/experimental effort through the design, implementation, and analysis of a special genetic retroactivity insulation device in yeast. This novel device effectively decouples transcriptional components that would otherwise be highly interlocked by allowing propagation of a regulatory signal in the forward direction while minimizing the undesired phenomena of retroactivity in the reverse direction. Besides providing an important new circuit element for synthetic biologists, the mathematical analysis of an insulating device will lead to an improved understanding of the extent to which modularity is present in regulatory networks in biological systems. The first aim of our research is to show that retroactivity affects regulatory networks without insulation and therefore that modularity is not a natural property of bio-molecular signaling pathways. Second, we will demonstrate a novel insulation device that counteracts retroactivity and allows a circuit to transmit information reliably despite loading from downstream clients. This special circuit will be designed and placed between two connected components to insulate them from retroactivity effects. We will study the device?s performance, ability to regulate many copies of a downstream component, and requirements for correct operation. Third, we will study how well the insulation device is decoupled from the cellular environment. To this end, we will perform system-level analysis and investigate crosstalk between the device and various important cellular processes. Intellectual Merit: Existing synthetic circuits lack an ability to insulate a driving input signal from retroactivity of the output load, precluding modular composition of complex biocircuits. To address this problem, we propose to construct and characterize a synthetic phosphorylation-based insulation device and instrumentation pathway that will demonstrate a general and modular technique for building sophisticated, large scale biological systems. Our novel technique leverages the integration of special rapid feedback mechanisms into biocircuits in order to create insulation devices, and hence has implications for the design and implementation of many other biological motifs and networks. Our research includes new theoretical and computational analysis of devices with feedback and retroactivity, and these new tools will also be applicable for the study of biological network problems other than retroactivity. This analysis is fundamentally important for tuning and characterizing desired insulation properties while minimizing interference with other cellular processes. Broader Impact: In synthetic biology, this research will lead to a general understanding of the engineering principles of modularity for bio-molecular systems design. Likewise, in systems biology, this research will address a fundamental question ? To what extent is modularity an inherent property of biological systems? The product of our collaborative efforts will lead to the discovery in natural systems of motifs similar to our insulation device and to the explanation of how modularity is achieved, including insight into when and where natural systems implement modularity and for what purposes. Ultimately, the resulting capability of modular composition to achieve defined engineering goals in biological systems will have tremendous impact on human therapeutics, including regenerative medicine, diabetes, and cancer therapy, as well as in other diverse areas, including biofuel production, environmental remediation, pharmaceutical production, and biosensing applications. This research will contribute to new interdisciplinary courses and will be integrated into a ten week undergraduate synthetic biology Summer program that culminates in an international competition and has a track record of attracting women, under-represented groups, and high schools students to the field.
