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物（MPC）？我将采用细菌双杂交和细菌三杂交试验来进一步研究GAC蛋白的相互作用。显微镜技术将用于分析每种蛋白质的共定位。为了了解每个GAC蛋白的化学计量学，同位素标记和交联质谱将被采用。这些数据将使我们第一次能够建立MPC模型。为阐明MPC（低温电子显微镜）内每个GAC蛋白的详细排列提供了一个起点。成熟的GAC如何从脂质转移到肽聚糖上？我的目标是利用生物信息学来鉴定LytR-Cps2A-Psr （LCP）家族的潜在功能和跨膜结构域。显微镜将用于可视化LCP蛋白相对于细胞膜的位置。分子克隆将用于镍层析拉下产物的缺失分析、放射性标记分析和Western blot分析。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