EAGER: Design of an RNA-based Dual Regulator for Repetitive Gene Expression Regulation

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The project integrates biological experiments and computational studies to train future scientists and engineers with competence in conducting interdisciplinary research, and to cultivate a multi-disciplinary perspective for problem-solving. Drawing expertise from engineering and biology, the project expands the state-of-the-art design of gene circuits with new capabilities that would enable cells to interact and respond in novel ways. This new means of programing cells with various capabilities will open doors to complex cellular designs with predictable and novel functionalities for applications in advanced biocomputing and medicine. The research also promotes participation of the K-12 and underrepresented students in STEM activities and advocates early STEM education and interdisciplinary research. These are critical elements for the success of the young generation scientists and engineers, as well as the sustainability and prosperity of future research. Current biological controllers are typically constrained by unidirectional and non-repetitive regulation of the target gene expression, which could hamper their applications in cases where repetitive up and down regulations are needed. The research plan involves mathematical modeling and control theory to guide the design of an RNA-based gene circuit and utilizes the in vitro transcription and translation (cell-free) system for rapid parts characterization and performance evaluation. Specifically, the project deploys an RNA-RNA annihilation reaction as the comparator between the reference and the target gene concentration. The outcome of the comparator then triggers the downstream up-regulator via CRISPRa (Clustered Regularly Interspaced Short Palindromic Repeats activation), or a down-regulator via RNA-RNA sequestration. By continuously evaluating the relative gene expression level and actuating the corresponding regulation pathway, repetitive bi-directional regulation is achieved. The project focuses on the design of genetic controllers with expanded regulation capability and improved feasibility for potential practical applications. In view of the interdisciplinary nature of the project, its findings are expected to benefit research in synthetic biology, mathematical modeling, control theory, and the cell-free systems.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.