Investigating glucose metabolism at the level of the individual bacterium

研究单个细菌水平的葡萄糖代谢

• 批准号: 1939247
• 金额: --
• 依托单位: UNIVERSITY OF EXETER
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2017
• 资助国家: 英国
• 起止时间: 2017 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-1939247
• 关键词: Investigating; glucose; metabolism; level; individual

The plasma membrane of biological cells acts as a barrier but also hosts a number of essential functions ensuring the viability of the cell. One of its main functions is the transport of various compounds across the membrane, e.g. uptake molecules essential for cell survival such as sugars, amino acids and ions and externalisation of molecules such as metabolic products and drugs. Transmembrane transport is usually carried out by special classes of transmembrane proteins, and it is paramount to understand how cells regulate the exchange of molecules in response to changes in the environment. This project will develop a novel approach based on model lipid bilayers, microfluidics and microscopy to characterise transport across membrane protein pores and understand the physical mechanisms underlying substrate uptake in living systems. Dr Pagliara's team recently used giant unilamellar vesicles (GUVs) and microfluidics to investigate molecular uptake through the outer membrane protein F, a transporter shown to play a role in bacterial antibiotic resistance (Cama et al. 2014, Lab Chip 14:2303; Cama et al. 2015, J Am Chem Soc 137:13836). Dr Petrov's team have a strong track record in investigating elasticity of cell and model membranes (including GUVs) and the role of the membrane physical properties in biological function. Prof Richards' team has optmised a protocol for purifying and tagging protein transporters to be reconstituted in GUVs. Building on this expertise, the student will obtain GUVs from lipids extracted from different microbial populations and reconstitute fluorescently labelled protein transporters in them. By engineering the geometry of bespoke microfluidic devices, the student will be able to trap GUVs embedded with transporters, deliver putative transported substrates and quantify substrate uptake and the number of reconstituted transporters via fluorescence microscopy. Quantitative image analysis and theoretical modeling (Pagliara et al. 2014, Phys Rev Lett, 113:048102; Locatelli et al. 2016, Phys Rev Lett, 117:038001) will allow determining substrate uptake kinetics, in terms of affinity, half-saturation constant and uptake rate, with single GUV resolution and comparing the different kinetics obtained for different combinations of protein transporters and GUVs. The effect of the reconstituted transporters on the mechanical properties of the membrane will be investigated using fluctuation spectroscopy and micropipette aspiration, techniques available in Petrov's laboratory.This project will help clarifying important biophysical questions related to the mechanisms of transmembrane transport in cells and could suggest novel approaches to designing antimicrobial drugs.

生物细胞的质膜充当屏障，但也具有确保细胞活力的许多基本功能。其主要功能之一是跨膜转运各种化合物，例如摄取细胞存活所必需的分子，如糖、氨基酸和离子，以及分子的外化，如代谢产物和药物。跨膜转运通常由特殊类型的跨膜蛋白进行，了解细胞如何调节分子交换以应对环境变化至关重要。 该项目将开发一种基于模型脂质双层，微流体和显微镜的新方法，以阻止跨膜蛋白孔的运输，并了解生命系统中底物摄取的物理机制。Pagliara博士的团队最近使用巨大的单层囊泡（GUV）和微流体来研究通过外膜蛋白F的分子摄取，外膜蛋白F是一种在细菌抗生素耐药性中起作用的转运蛋白（Cama等人，2014，Lab Chip 14：2303; Cama等人，2015，J Am Chem Soc 137：13836）。彼得罗夫博士的团队在研究细胞和模型膜（包括GUV）的弹性以及膜物理特性在生物功能中的作用方面有着良好的记录。理查兹教授的团队已经选择了一种纯化和标记蛋白质转运蛋白的方案，以便在GUV中重建。在此专业知识的基础上，学生将从不同微生物种群中提取的脂质中获得GUV，并在其中重建荧光标记的蛋白质转运蛋白。通过设计定制微流体装置的几何形状，学生将能够捕获嵌入转运蛋白的GUV，递送推定的转运底物，并通过荧光显微镜量化底物吸收和重组转运蛋白的数量。定量图像分析和理论建模（Pagliara等人，2014，Phys Rev Lett，113：048102; Locatelli等人，2016，Phys Rev Lett，117：038001）将允许在亲和力、半饱和常数和摄取速率方面确定底物摄取动力学，采用单一GUV分辨率，并比较蛋白转运蛋白和GUV的不同组合获得的不同动力学。重组转运蛋白对膜机械性能的影响将使用波动光谱和微量吸管抽吸技术进行研究，这些技术可在Petrov的实验室中使用。该项目将有助于澄清与细胞跨膜转运机制相关的重要生物物理问题，并可能为设计抗微生物药物提供新的方法。