Mechanisms of a Natural Bacterial Biosensor Using RT-DNA

该项目旨在确定被称为逆转录的细菌免疫系统如何保护细胞群体免受病毒侵害。反转录子是一种独特的免疫系统，人们对其了解甚少，它使用反转录的DNA来感知细菌病毒并引发免疫反应。本计画将使用定量的方法来深入了解retron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（例如我们自己的人类微生物组）以及利用细菌的工业过程的理解中的关键空白。Retron组件在生物制造和生物医学中也具有重要的应用潜力。因此，本项目中retron的基础研究将为未来先进生物技术的发展提供信息。这项研究的更广泛影响包括为从两年制和四年制大学过渡的代表性不足的学生提供培训，并与这些学生合作，使用3D打印部件和简单的DIY电子产品开发retron发现套件。以及计划和协议的任何人谁想要建立这个作为一个实验或课程计划。这项工作的中心问题是如何产生的DNA片段，细胞可以作为噬菌体生物传感器的关键元件。工作将通过平行调查（1）逆转录DNA的序列和结构，（2）retron的蛋白质相互作用组，（3）在天然宿主中retron表达的群体动力学。所有这些研究都将使用定量的高通量方法，包括合成变体文库、定量质谱和多重单细胞分析。完成后，这些实验将回答（1）逆转录的DNA如何与retron的蛋白质组分相互作用，以在没有噬菌体的情况下中和它们，（2）这些蛋白质组分如何连接到细胞的更广泛的蛋白质组中以实现生物反应，以及（3）细胞群体如何平衡使用自-该奖项反映了NSF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.