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

这项研究的目的是开发新的代谢物传感和代谢物诱导的酶定位的策略；这将有助于基础细胞知识，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成能够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实现的，该网络严格调节细胞成分的活动，使其达到所需的水平，以适应不断变化的营养条件。对这些代谢物的详细分析将有助于更深入地了解它们在促进和调节细胞过程中的生理作用。为了实现这一点，该研究利用分裂的RNA适配子片段的可逆重组来实时检测代谢物。同样的可逆分裂适配子组装设计也被用来创建动态代谢素，以揭示对酵母代谢的洞察，从而优化产品合成。开发的工具集可以很容易地转移到其他真核生物，如哺乳动物细胞，以解决有关新陈代谢的调节和重新连接的基本问题。这项研究横跨生物、化学和工程学等核心学科，为学生在各个层次和多个领域提供了充足的培训机会。该项目还通过特拉华大学和加州大学欧文分校现有的项目，促进了与当地高中教师和学生的外联活动。细胞内代谢物的实时量化对于我们询问、理解和设计一系列生物系统中的新陈代谢是至关重要的。该项目利用分裂的RNA适配子片段的可逆重组作为实时代谢物检测的新框架。使用Cas6介导的蛋白质-RNA组装策略，使用分裂荧光蛋白报告的可逆组装来监测代谢物诱导的分裂RNA适配子重组的实时探测。分裂适配子组装的可逆性也被用来产生动态代谢物，用于代谢反应控制，这对广泛的基础研究和合成生物学应用是有用的。这项研究通过创建一种在许多感兴趣的生物体中实时检测代谢物和代谢物介导的代谢物动态组装的新方法，影响了合成生物学领域。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.