Mechanisms of a Natural Bacterial Biosensor Using RT-DNA

该项目旨在确定被称为逆转录酶的细菌免疫系统如何保护细胞群免受病毒的侵害。逆转录酶是一种独特且鲜为人知的免疫系统，它利用逆转录DNA来感知细菌病毒并引发免疫反应。该项目将使用定量方法来深入了解逆转录系统的分子机制。所获得的知识将填补我们对自然微生物生态系统的理解的关键空白，例如我们自己的人类微生物组，以及利用细菌的工业过程。逆转录成分在生物制造和生物医学方面也具有重要的应用潜力。因此，本项目对逆转录酶的基础研究将为未来先进生物技术的发展提供信息。这项研究的广泛影响包括对代表性不足的学生进行培训，从两年制和四年制大学过渡，并与这些学生一起使用3D打印部件和简单的diy电子产品开发逆冲发现套件，以及为任何想要将其构建为实验或课程计划的人制定计划和协议。这项工作的核心问题是细胞产生的DNA片段如何作为噬菌体生物传感器的关键元素。工作将通过平行研究(1)逆转录DNA的序列和结构，(2)逆转录子的蛋白质相互作用组，以及(3)逆转录子在原生宿主中表达的种群动态进行。所有这些研究都将使用定量、高通量的方法，包括合成变异文库、定量质谱分析和多路单细胞分析。完成后，这些实验将回答(1)逆转录的DNA如何与逆转录的蛋白质成分相互作用以在缺乏噬菌体的情况下中和它们，(2)这些蛋白质成分如何与细胞更广泛的蛋白质组连接以产生生物反应，以及(3)细胞群体如何平衡使用自毒系统抵御噬菌体感染的成本和收益。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。

This project aims to determine how bacterial immune systems known as retrons defend cell populations against viruses. Retrons are unique, and poorly understood, immune systems that use reverse-transcribed DNA to sense bacterial viruses and set off an immune response. This project will use quantitative approaches to gain insight into the molecular mechanisms of retron systems. The knowledge gained will fill in critical gaps in our understanding of natural microbial ecosystems, such as our own human microbiome, in addition to industrial processes that leverage bacteria. Retron components also hold important potential for use in biomanufacturing and biomedicine. Thus, the fundamental studies of the retron in this project will inform future development of advanced biotechnologies. The Broader Impacts of this research include training for underrepresented students transitioning from two and four year colleges and working with those students to develop retron discovery kits using 3D printed parts and simple, do-it-yourself electronics, as well as the plans and protocols for anyone who wants to build this as an experiment or lesson plan.The central question in this work is how a fragment of DNA that is produced by cells can serve as the key element of a phage biosensor. Work will progress through parallel investigations into (1) the sequence and structure of the reverse transcribed DNA, (2) the protein interactome of the retron, and (3) the population dynamics of retron expression in a native host. All of these investigations will use quantitative, high-throughput approaches, including synthesized variant libraries, quantitative mass spectrometry, and multiplexed single cell analysis. When completed, these experiments will answer (1) how the reverse transcribed DNA interacts with the protein components of the retron to neutralize them in the absence of phage, (2) how those protein components link into the broader proteome of the cell to effect a biological response, and (3) how a population of cells balances the costs and benefits of using a self-toxic system to defend against phage infection.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.