IN VIVO STUDY OF TRANSCRIPTIONAL REGULATION IN BACILLI BY FCS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 本计画提出利用最先进的双光子萤光相关光谱技术，直接在活体内观察枯草芽孢杆菌的转录调控。 该项目涉及两个转录的研究 来自B的阻遏物。subtilis、CggR和CcpN。 自从CggR和CcpN在B.枯草芽孢杆菌基因组（Kunst等，1997）。 这两种转录抑制因子通过产生代谢酶控制革兰氏阳性菌碳代谢循环的相反方向。 我们建议在单分子水平上直接观察体内转录调控。 利用B.本研究利用点扫描双光子荧光相关光谱（FCS和sFCS）、数量和亮度（N&B）和光栅图像相关光谱（RICS）技术，对枯草芽孢杆菌（Bacillus subtilis）进行了荧光分析。 我们将阐明在体外研究中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.