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

该项目将生物实验与计算研究相结合，以培养具有跨学科研究能力的未来科学家和工程师，并培养解决问题的多学科视角。利用工程学和生物学的专业知识，该项目扩展了最先进的基因电路设计，使细胞能够以新颖的方式相互作用和反应。这种对具有各种功能的细胞进行编程的新方法将为具有可预测和新颖功能的复杂细胞设计打开大门，用于先进的生物计算和医学应用。该研究还促进了K-12和代表性不足的学生参与STEM活动，并倡导早期STEM教育和跨学科研究。这些都是年轻一代科学家和工程师成功的关键因素，也是未来研究的可持续性和繁荣的关键因素。目前的生物控制器通常受到目标基因表达的单向和非重复调控的限制，这可能会阻碍它们在需要重复上下调控的情况下的应用。该研究计划涉及数学建模和控制理论来指导基于rna的基因电路的设计，并利用体外转录和翻译（无细胞）系统进行快速部件表征和性能评估。具体来说，该项目部署了一个RNA-RNA湮灭反应作为参比和靶基因浓度之间的比较。然后比较器的结果通过CRISPRa （Clustered Regularly Interspaced Short Palindromic Repeats activation）触发下游的上调调控，或通过RNA-RNA隔离触发下调调控。通过不断评估相关基因表达水平，启动相应的调控通路，实现重复的双向调控。该项目侧重于设计具有扩展调节能力和提高潜在实际应用可行性的遗传控制器。鉴于该项目的跨学科性质，其研究结果有望有益于合成生物学、数学建模、控制理论和无细胞系统的研究。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

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.