Investigating glucose metabolism at the level of the individual bacterium

生物细胞的质膜起着屏障的作用，但也具有许多基本功能，确保细胞的生存能力。它的主要功能之一是跨膜运输各种化合物，例如摄取糖、氨基酸和离子等细胞生存所必需的分子，以及代谢产物和药物等分子的外化。跨膜运输通常是由特殊类型的跨膜蛋白完成的，了解细胞如何调节分子交换以响应环境的变化是至关重要的。该项目将开发一种新的方法，基于模型脂双层、微流体和显微镜来表征跨膜蛋白孔的运输，并了解生物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，F是一种被证明在细菌抗生素耐药性中发挥作用的转运蛋白(Cama等人)。2014，实验室芯片14：2303；Cama等人。2015年，Jam 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)的弹性以及膜的物理性质在生物功能中的作用方面有着良好的记录。理查兹教授的团队选择了一种用于纯化和标记蛋白质转运体的协议，该协议将在GUV中重组。在此专业知识的基础上，学生将从从不同微生物种群中提取的脂类中获得GUV，并在其中重建荧光标记的蛋白质转运体。通过设计定制的微流体设备的几何结构，学生将能够捕获嵌入转运体的GUV，提供假定的转运体底物，并通过荧光显微镜定量底物摄取和重组转运体的数量。定量图像分析和理论建模(Pagliara等人)。2014年，Phys Rev Lett，113：048102；Locatelli等人。2016年，Phys Rev Lett，117：038001)将允许用单一GV分辨率确定底物吸收动力学，包括亲和力、半饱和常数和摄取速率，并比较不同蛋白质转运体和GUV组合获得的不同动力学。重组的转运体对膜的机械性能的影响将使用波动光谱和微管吸气技术来研究，这是彼得罗夫实验室可用的技术。这个项目将有助于澄清与细胞内跨膜运输机制相关的重要生物物理问题，并可能为设计抗菌药物提供新的方法。

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.