Insight into the GAC biosynthesis machinery and how GAC is linked to Peptidoglycan in S. pyogenes cells.

GAC蛋白是否形成多蛋白复合体(MPC)？我将使用细菌双杂交和细菌三杂交实验进一步研究GAC蛋白之间的相互作用。显微技术将被用来分析每种蛋白质的共定位。为了了解每种GAC蛋白的化学计量比，将使用同位素标记和交联质谱。这些数据将使我们第一次能够建立MPC模型。为阐明每个GAC蛋白在MPC(低温电子显微镜)中的详细排列提供了一个起点。成熟的GAC是如何从脂类转移到肽聚糖上的？我的目标是利用生物信息学来确定LytR-Cps2A-PSR(LCP)家族潜在的功能和跨膜结构域。显微镜将被用来显示LCP蛋白相对于细胞膜的位置。分子克隆将用于对镍层析下拉产品进行缺失分析、放射性标记分析和蛋白质印迹分析。该项目提供跨学科技能培训

Are GAC proteins forming a multi-protein complex (MPC)?I will employ bacterial two-hybrid and bacterial three-hybrid assays to further investigate interactions of GAC proteins. Microscopy techniques will be utilised to analyse colocalisation of each protein. To understand the stoichiometry of each GAC protein, isotope labelling and cross-linking mass spectrometry will be employed. This data will allow us, for the first time, to build a MPC model. Providing a starting point to elucidate the detailed arrangement of each GAC protein within the MPC (cryogenic electron microscopy).How is the mature GAC transferred from the lipid onto peptidoglycan?I aim to employ bioinformatics to identify potential functional and transmembrane domains of the LytR-Cps2A-Psr (LCP) family. Microscopy will be utilised to visualise the location of LCP proteins in relation to the cell membrane. Molecular cloning will be used to perform deletion assays, radiolabelling assays and Western blot analysis on nickel chromatography pull-down products.Project provides training in Interdisciplinary Skills