Collaborative Research: NSF/MCB: Repurposing metabolite-responsive aptamers for real-time sensing and dynamic control of Cas6-mediated metabolon assembly

合作研究：NSF/MCB：重新利用代谢物响应适体，用于 Cas6 介导的代谢物组装的实时传感和动态控制

• 批准号: 2317399
• 负责人: Nancy Da Silva
• 金额: $ 59万
• 依托单位: University of California-Irvine
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-15 至 2026-07-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2317399&HistoricalAwards=false
• 关键词: Collaborative; Research; NSF; MCB; Repurposing

The aim of the research is to develop novel strategies for metabolite sensing and metabolite-induced enzyme localization; this will contribute to fundamental cellular knowledge and improve the efficiency of bioprocesses that are associated with synthetic biology. In nature, many microorganisms have evolved to survive across different growth-permissive conditions. This adaptability is achieved through a highly coordinated metabolic network that tightly regulates the activity of cellular components at the required level in order to adjust to fluctuating nutrient conditions. A detailed analysis of these metabolites would provide a deeper understanding of their physiological roles in promoting and regulating cellular processes. To achieve this, the research exploits the reversible reconstitution of split RNA aptamer fragments for real-time metabolite sensing. The same reversible split aptamer assembly design is also exploited to create dynamic metabolons to reveal insights into yeast metabolism for optimizing product synthesis. The tool sets developed are easily transferrable to other eukaryotes such as mammalian cells to address fundamental questions about regulation and rewiring of metabolism. The research spans the core disciplines of biology, chemistry, and engineering, in providing ample opportunities for student training at all levels and in multiple areas. This project also facilitates outreach activities to local high school teachers and students through existing programs available at the University of Delaware and UC Irvine.Real-time quantification of intracellular metabolites is essential for our ability to interrogate, understand, and engineer metabolism in a range of biological systems. This project exploits the reversible reconstitution of split RNA aptamer fragments as a new framework for real-time metabolite sensing. Using a Cas6-mediated protein-RNA assembly strategy, real-time probing of metabolite-induced split RNA aptamer reconstitution is monitored using the reversible assembly of a split fluorescent protein reporter. The reversible nature of split aptamer assembly is also exploited to create dynamic metabolons for metabolite-responsive control of metabolism that is useful for a wide range of fundamental studies and synthetic biology applications. This research impacts the field of synthetic biology by creating a new method for real-time metabolite sensing and for metabolite-mediated dynamic assembly of metabolons in many organisms of interest.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.

该研究的目的是制定代谢物传感和代谢产物引起的酶定位的新策略。这将有助于基本的细胞知识，并提高与合成生物学相关的生物过程的效率。在自然界中，许多微生物已经进化为在不同的生长允许条件下生存。这种适应性是通过高度协调的代谢网络来实现的，该网络严格调节所需水平的细胞成分的活性，以适应养分的波动。对这些代谢产物的详细分析将对它们在促进和调节细胞过程中的生理作用有更深入的了解。为了实现这一目标，研究利用了分裂RNA适体片段的可逆重构来实时代谢物传感。还利用了相同的可逆分裂适体装配设计来创建动态代谢物，以揭示对酵母代谢的见解，以优化产品合成。开发的工具集很容易转移到其他真核生物，例如哺乳动物细胞，以解决有关代谢调节和重新布线的基本问题。该研究涵盖了生物学，化学和工程学的核心学科，为在各个级别和多个领域提供了充足的学生培训机会。该项目还通过特拉华大学和UC Irvine的现有计划为当地的高中教师和学生提供了外展活动。对细胞内代谢物的真实时间量化对于我们在一系列生物系统中询问，理解和工程代谢的能力至关重要。该项目利用了分裂RNA适体片段的可逆重构，作为实时代谢物传感的新框架。 使用CAS6介导的蛋白-RNA组装策略，使用分裂荧光蛋白报告基因的可逆组装来​​监测代谢物诱导的分裂RNA适体的实时探测。还利用了分裂适体组件的可逆性，以创建动态代谢物，用于代谢物对代谢的反应性控制，这对于广泛的基本研究和合成生物学应用非常有用。这项研究通过创建一种新的代谢物传感的方法来影响合成生物学领域，并在许多感兴趣的生物体中代谢物介导的代谢物的动态组装。该奖项反映了NSF的法定任务，并被认为是通过基金会的知识分子和更广泛的影响来评估的支持，并被认为是值得的。