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

BBSRC-NSF/BIO：TXTL 合成细胞系统中膜蛋白组装的实时纳米传感

• 批准号: 2017932
• 负责人: Vincent Noireaux
• 金额: $ 66.68万
• 依托单位: University of Minnesota-Twin Cities
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2017932&HistoricalAwards=false
• 关键词: BBSRC; NSF; BIO; Real; time

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.

模拟特定生物功能的合成细胞的自下而上的构建具有解决与人类健康和环境相关的某些社会问题的潜力。目前最先进的合成细胞生产中的一个严重限制是缺乏如何将特定的膜相关功能工程化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-蛋白质相互作用和动力学的基本方面的定量理解。这个合作项目的目标是使用尖端的传感器技术来推进我们对遗传编程合成细胞中膜蛋白功能的基本理解。 该项目是明尼苏达大学双城分校（美国）和埃克塞特大学（英国）之间的合作，旨在为早期职业科学家提供跨学科研究的机会，并将尖端生物技术引入本科生的课堂。 这个跨学科的项目将提供新的定量信息的各种动态功能的膜蛋白及其自组装，通过应用高灵敏度的回音壁模式的单分子传感器，以一套三个生物膜功能概括在一个合成的细胞系统：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进行验证，以提供关于大尺度和膜蛋白在脂质双层的集体效应的补充信息。研究工作分为三个目标，将在三年项目期间进行。首先，将由装载有无细胞表达反应以表达膜蛋白的脂质体组成的合成细胞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，我们将使用回音壁模式传感器和微量天平监测以前无法访问的细胞骨架蛋白，介导细胞的形状和分裂在合成细胞的膜的吸附动力学，作为脂质膜特性的函数。第三，单分子实验将通过几个独立的传感通道进行，以表征双组分系统信号传导期间的真实的时间生物分子结构变化。纳秒到小时的传感器通道的检测时间尺度将提供信息，以分析蛋白质运动的层次结构在两个组件系统的信号，相对于物理和化学刺激。微量天平将提供关于膜的生物物理和生物化学性质与双组分系统的蛋白质之间的相互作用之间的关系的信息。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。英国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理事会支持。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。

项目成果

Membrane functions genetically programmed in synthetic cells: A barrier to conquer

• DOI: 10.1016/j.coisb.2020.09.006
• 发表时间: 2020-09
• 期刊: Current Opinion in Systems Biology
• 影响因子: 3.7
• 作者: David Garenne;V. Noireaux