IN VIVO STUDY OF TRANSCRIPTIONAL REGULATION IN BACILLI BY FCS

FCS对杆菌转录调控的体内研究

• 批准号: 8171006
• 负责人: CATHERINE A ROYER
• 金额: $ 0.47万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-01 至 2011-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171006
• 关键词: Arts; Bacillus (bacterium); Bacillus subtilis; Carbon; Chimeric Proteins; Computer Retrieval of Information on Scientific Projects Database; Engineering; Enzymes; Fluorescence; Funding; Genome; Gram-Positive Bacteria; Grant; Image; In Vitro; Institution; Investigation; Metabolic; Production; Research; Research Personnel; Resources; Scanning; Source; Spectrum Analysis; Techniques; Transcription Repressor/Corepressor; Transcriptional Regulation; United States National Institutes of Health; Work; in vivo; promoter; single molecule; two-photon

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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目和 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 该项目提出使用最先进的双光子荧光相关光谱法通过直接体内观察来研究枯草芽孢杆菌的转录调控。 该项目涉及两种转录的研究 来自枯草芽孢杆菌、CggR 和 CcpN 的阻遏蛋白。 自从在枯草芽孢杆菌基因组测序中发现 CggR 和 CcpN（Kunst 等，1997）以来，已经在体外对 CggR 和 CcpN 进行了大量工作（Zorrilla 等，2007a；Zorrilla 等，2007b；Zorrilla 等，2008a；Zorrilla 等，2008b）。 两种转录抑制因子通过产生代谢酶来控制革兰氏阳性细菌中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。 利用枯草芽孢杆菌的基因工程菌株，在天然启动子下含有与 CggR、CcpN 和相关因子融合的荧光蛋白，我们将应用点扫描二光子荧光相关光谱（FCS 和 sFCS）、数量和亮度（N&B）以及光栅图像相关光谱（RICS）技术。 我们将阐明体外研究无法观察到的转录调控机制。