This application 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) and raster image correlation spectroscopy (RICS). We will elucidate mechanisms of transcriptional regulation that cannot be observed by in vitro investigation.
Field of science
- /natural sciences/chemical sciences/analytical chemistry/spectroscopy
- /natural sciences/biological sciences/genetics and heredity/genome
- /natural sciences/biological sciences/biochemistry/biomolecules/proteins/enzymes
Call for proposal
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