Resolving the Mechano-Chemical Regulation of Microbial Populations in Microfluidic Devices

 描述（由申请人提供）：当细胞生长和分裂形成密集群体时，它们会发生化学和物理相互作用。例如，生长中的肿瘤或微生物/真菌生物膜中的细胞竞争营养和空间，从而相互施加化学和物理压力。虽然近年来已经发现了一个以前隐藏的层的命运决定和哺乳动物组织中的生长速率的机械调节，很少有人知道机械约束单细胞微生物的后果，主要是由于缺乏适当的培养技术。拟议研究的目的是通过量化空间受限的微生物群落对明确定义的化学和物理应力的细胞和多细胞反应来填补这一空白。为此，PI提出了严格控制的微流体实验和新颖的生物物理模拟和理论，弥合了单细胞和整个群体之间时空尺度上的差距。拟议的研究利用理论和实验之间的持续反馈，根据单个细胞的联合行动，实现对微生物种群自组织的预测性理解。这些结果将大大推进我们对生物膜形成的时空方面的理解，并具体阐明细胞群体如何对物理和化学线索的组合做出反应，这是合理设计对抗微生物和真菌生物膜生长并限制其进化耐药性的策略的关键。此外，计划中的新型微流体装置和计算机模拟将对生物物理界具有广泛的实用性，以实现解剖微生物种群的集体特性的目标。私家侦探有三个具体目标。首先，他将开发一种新的微流体培养装置设计，即微流体机械恒化器，其中化学和机械条件可以严格控制。其次，他将使用该设备结合生物物理建模来探索细胞对机械化学信号的反应，首先关注单细胞真菌和细菌。第三，从微流体群体测量中推断，他将开发理论和模拟来预测单个细胞联合作用的群体行为。Aim 1使用最先进的微流体技术来超越缺乏物理控制的微流体培养装置的局限性。Aim 2的实验方法是基于微流体室中细胞的自动时空跟踪和报告基因表达变化的荧光标记。为目标3开发的模拟将现代种群生物学理论与物理和化学领域的分子动力学相结合。

 DESCRIPTION (provided by applicant): When cells grow and divide to form dense populations, they interact both chemically and physically. For instance, cells in growing tumors or microbial/fungal biofilms compete for nutrients and space, thereby exerting chemical and physical stresses on each other. Although recent years have uncovered a previously hidden layer of mechanical regulation of fate determination and growth rates in mammalian tissues, little is known about the consequences of mechanical constraints on single-celled microbes, largely, due to a lack of appropriate culturing techniques. The objective of the proposed research is to fill this gap by quantifying the cellular and multi- cellular response of spatially confined microbial communities to well-defined chemical and physical stresses. To this end, the P.I. proposes tightly-controlled microfluidic experiments and novel biophysical simulations and theory that bridges the gap in spatio-temporal scales between single cells and entire populations. The proposed research leverages a continual feedback between theory and experiments to achieve a predictive understanding of self-organization in microbial populations in terms of the joint actions of individual cells. The results will significantly advance our understanding of spatio-temporal aspects of biofilm formation, and elucidate specifically how cellular populations respond to combinations of physical and chemical cues, which is key to the rational design of strategies to battle microbial and fungal biofilm growth and to limit their abilty to evolve drug resistance. Further, the planned novel microfluidic devices and computer simulations will be of broad utility to the biophysics community for the goal of dissecting collective properties of microbial populations. The P.I. has three specific aims. First, he will develop a novel design for microfluidic culturing devices, a microfluidic mechano-chemostat, in which chemical and mechanical conditions can be tightly controlled. Second, he will use this device in conjunction with biophysical modeling to explore cellular response to mechano-chemical cues, focusing at first on single-celled funghi and bacteria. Third, extrapolating from microfluidic population measurements, he will develop theory and simulations to predict the behavior of populations from the joint action of individual cells. Aim 1 uses state-of-the-art microfluidic techniques to transcend the limitations of microfluidic culturing devices, which lack physical control. The experimental approaches to Aim 2 are based on automated spatio-temporal tracking of cells in microfluidic chambers and fluorescence markers reporting changes in gene expression. The simulations developed for Aim 3 synthesize modern population biology theory with the molecular dynamics of physical and chemical fields.