Deriving biological principles from replication initiation control in bacteria

该项目旨在阐明细菌细胞如何决定何时开始DNA复制，并传播研究中使用的关键单细胞技术。虽然DNA复制是细胞繁殖最精确的过程之一，但尽管进行了广泛的研究，但细胞如何在不同的生长条件下保持精度50多年来一直没有解决。为此，该项目汇集了来自多个学科的工具和思维方式，包括允许连续跟踪大量单个细胞的单细胞技术。这些技术最初是在PI的实验室发明的，多年来得到了简化，即使是本科生也可以与研究生和博士后研究人员一起进行原创研究。在拟议的活动中，来自未被充分代表的少数群体的本科生将利用现代知识传播平台向广泛的科学界传播单细胞技术，以便一个典型的生物实验室几乎可以免费实施相同的方法。一些关键实验一旦完成，将被纳入PI将开发的生物物理实验室课程的设计中。研究计划将集中在DNAA上，这是一种广泛保守的细菌复制起始的主调控因子。基于启动时细胞大小对不同生长条件的响应，提出了一个定量模型来解释DNAA的产生及其生化特性如何以一种独立于生理的方式强有力地控制启动过程。该模型的预测将通过使用DNAA和相关蛋白质的遗传突变，以及使用最先进的微流体和成像方法对各种单细胞参数进行精确测量来验证。这项研究的最终结果将提供对DNA复制启动的精确控制的机械洞察，可概括为广泛的生理条件。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 project aims to elucidate how bacterial cells decide when to start DNA replication, and also disseminate key single-cell technologies used in the research. While DNA replication is one of the most precise processes underlying cellular reproduction, how cells maintain the precision under different growth conditions has remained unsolved for over 50 years despite extensive studies. To this end, the project brings together tools and ways of thinking from multiple disciplines, including single-cell technologies that allow continuous tracking of a large number of individual cells. These technologies were originally invented in the PI’s laboratory, and have been streamlined over the years so that even undergraduate students can perform original research alongside graduate students and postdoctoral researchers. In the proposed activities, undergraduate students from underrepresented minority groups will disseminate the single- cell technologies to the broad scientific community using modern knowledge dissemination platforms, so that a typical biological lab can implement the same approaches practically at no cost. Some of the key experiments, once completed, will be integrated into the design of a biological physics laboratory course that the PI will develop.The research program will focus on DnaA, a widely conserved master regulator of replication initiation in bacteria. Based on how cell size at initiation responds to various growth conditions, a quantitative model is proposed to explain how production of DnaA and their biochemical properties robustly control the initiation process in a physiology-independent manner. The predictions of the model will be tested by using genetic mutants of DnaA and associated proteins, as well as precision measurements of various single-cell parameters using state-of-the-art microfluidics and imaging methods. The final output of this research will provide a mechanistic insight into the precision control of DNA replication initiation generalizable to a wide range of physiological conditions.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.