IN VIVO STUDY OF TRANSCRIPTIONAL REGULATION IN BACILLI BY FCS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 该项目提出了通过使用最先进的双光子荧光相关光谱直接在体内观察来研究枯草芽孢杆菌的转录调控。该项目涉及对两个转录的 枯草杆菌、CggR和CcpN的抑制子。自从在枯草杆菌基因组测序中发现CggR和CcpN(Zorrilla等人，2007a；Zorrilla等人，2007b；Zorrilla等人，2008a；Zorrilla等人，2008b)以来，在体外已经做了大量的工作(Kost等人，1997年)。在革兰氏阳性细菌中，这两种转录抑制物通过产生代谢酶来控制碳代谢循环中的相反方向。我们建议在单分子水平上直接观察体内转录调控。利用含有荧光蛋白的枯草杆菌基因工程菌株，在天然启动子的作用下，融合了CggR、CcpN及相关因子，我们将应用点扫描双光子荧光相关光谱(FCS和SFCS)、数量和亮度(N&B)和栅格图像相关光谱(RICS)技术。我们将阐明体外研究无法观察到的转录调控机制。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This project proposes the study of transcriptional regulation in Bacillus subtilis by direct, in vivo, observation using state-of-the-art two-photon fluorescence correlation spectroscopy. The project involves the study of two transcriptional repressors from B. subtilis, CggR and CcpN. Much work has been done in vitro on CggR and CcpN (Zorrilla et al, 2007a; Zorrilla et al, 2007b; Zorrilla et al, 2008a; Zorrilla et al, 2008b) since their discovery in the sequencing of the B. subtilis genome(Kunst et al, 1997). Both transcriptional repressors control opposite directions in the carbon metabolic cycle in gram positive bacteria through the production of metabolic enzymes. We propose to directly observe transcriptional regulation in vivo at the single molecule level. Using genetically engineered strains of B. subtilis, containing fluorescent protein fusions with CggR, CcpN and related factors under native promoters, we will apply the techniques of point and scanning two photon fluorescence correlation spectroscopy (FCS and sFCS), Number and Brightness (N&B) and raster image correlation spectroscopy (RICS). We will elucidate mechanisms of transcriptional regulation that cannot be observed by in vitro investigation.