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 复制是细胞复制中最精确的过程之一，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确度的问题在 50 多年来仍然没有得到解决。为此，该项目汇集了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 的实验室中发明的，多年来经过不断改进，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，来自代表性不足的少数群体的本科生将利用现代知识传播平台向广大科学界传播单细胞技术，以便典型的生物实验室实际上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。该研究计划将重点关注 DnaA，一种广泛保守的细菌复制启动主调节因子。基于起始时的细胞大小如何响应各种生长条件，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独立于生理学的方式稳健地控制起始过程。该模型的预测将通过使用 DnaA 和相关蛋白质的基因突变体以及使用最先进的微流体和成像方法精确测量各种单细胞参数进行测试。这项研究的最终成果将为 DNA 复制起始的精确控制提供一个机制见解，可推广到广泛的生理条件。该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值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.