BBSRC-NSF/BIO: Real-time Nanosensing of Membrane Protein Assembly in a TXTL Synthetic Cell System

模拟特定生物功能的自下而上的合成细胞结构有可能解决与人类健康和环境有关的某些社会问题。目前合成细胞生产的一个严重限制是缺乏关于如何将特定的膜相关功能设计到细胞中的基本知识。事实上，将膜功能整合到合成细胞中需要对膜-蛋白相互作用和动力学的基本方面有定量的了解。这个合作项目的目标是利用尖端的传感器技术，推进我们对基因编程合成细胞中膜蛋白功能的基本理解。这个项目是明尼苏达大学双城分校（美国）和埃克塞特大学（英国）的合作项目，也旨在为早期职业科学家提供进行跨学科研究的机会，并将尖端生物技术引入本科学生的课堂。这个跨学科的项目将提供关于膜蛋白的各种动态特征及其自组装的新的定量信息，通过将高灵敏度的窃窃廊模式单分子传感器应用于合成细胞系统中再现的三种生物膜功能：膜通道、细胞骨架和双组分信号转导系统。单分子实验将通过石英晶体微天平与耗散方法并行，以提供大尺度和脂质双分子层膜蛋白集体效应的补充信息。研究工作分为三个目标，将在三年的项目中进行。首先，合成细胞由脂质体组成，脂质体装载无细胞表达反应来表达膜蛋白，将其设置在单分子传感器上，以监测天然膜蛋白通道的插入并表征其活性。其次，我们将使用窃窃廊模式传感器和微天平来监测细胞骨架蛋白在合成细胞膜上介导细胞形状和分裂的吸附动力学，作为脂质膜特性的功能。第三，单分子实验将通过几个独立的传感通道进行，以实时表征双组分系统信号传递过程中的生物分子结构变化。传感器通道的纳秒到小时的检测时间尺度将提供信息来分析两个组成系统信号中蛋白质运动的层次结构，以及物理和化学刺激。微天平将提供膜生物物理和生物化学特性之间的关系以及双组分系统中蛋白质之间的相互作用的信息。该项目将支持本科生和博士后研究人员的跨学科培训，项目的各个方面将纳入本科生课程的课程。这个英美合作项目由美国国家科学基金会和英国生物技术和生物科学研究委员会支持。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。

The bottom-up construction of synthetic cells that emulate specific biological functions has the potential to address certain societal problems related to human health and the environment. A serious limitation in the current state-of-the-art production of synthetic cells is the lack of basic knowledge of how particular membrane-associated functions can be engineered into the cells. Indeed, integrating membrane functions into synthetic cells requires a quantitative understanding of basic aspects of membrane-protein interactions and dynamics. The goal of this collaborative project is to advance our fundamental understanding of membrane protein functions in genetically programmed synthetic cells using a cutting-edge sensor technology. This project, which is a collaboration between the University of Minnesota-Twin Cities (US) and the University of Exeter (UK), also aims to provide early career scientists with opportunities to do interdisciplinary research and to introduce cutting-edge biotechnology into the classroom of undergraduate students. This interdisciplinary project will provide novel quantitative information on various dynamic features of membrane proteins and their self-assembly by applying highly-sensitive whispering gallery mode single-molecule sensors to a set of three biological membrane functions recapitulated in a synthetic cell system: membrane channels, cytoskeleton and two-component signal transduction systems. The single molecule experiments will be paralleled by a quartz crystal microbalance with dissipation approach to provide complementary information on large scales and collective effects of membrane proteins at the lipid bilayer. The research effort is divided into three objectives, to be carried out during the three-year project. First, the synthetic cell, consisting of a liposome loaded with a cell-free expression reaction to express the membrane proteins, will be set up on the single molecule sensor to monitor the insertion of native membrane protein channels and to characterize their activity. Second, we will use the whispering gallery mode sensors and the microbalance to monitor previously inaccessible adsorption kinetics of cytoskeletal proteins that mediate cell shape and division at the membrane of synthetic cell, as a function of lipid membrane properties. Third, the single molecule experiments will be performed by several independent sensing channels to characterize in real time biomolecular structural changes during signaling of a two-component system. Nanoseconds to hour’s detection timescales of sensor channels will provide information to analyze the hierarchy of protein motions in two component systems signaling, with respect to physical and chemical stimuli. The microbalance will provide information on the relationship between the membrane biophysical and biochemical properties of the membrane and the interaction between the proteins of the two-component system. This project will support the interdisciplinary training of undergraduates and post-doctoral researchers and aspects of the project will be integrated into curriculum of an undergraduate student course.This collaborative US/UK project is supported by the US National Science Foundation and the UK Biotechnology and Biological Sciences Research Council.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.