Start Date: Aug 01, 2010
End Date: Sep 30, 2014
We will obtain genomic DNA, mRNA, and protein preparations from C. thermocellum and other CBP bacteria grown on different substrates and/or at different growth rates. The mRNA will be used for microarray analysis of gene expression, and the protein preparations will be used for proteomic identification and quantitation of specific proteins involved in polysaccharide degradation. DNA will be used to PCR-amplify genes of interest (determined from microarray and proteomic experiments) or for site-directed mutagenesis. These DNA preparations will be used in an in vitro transcription/translation system to produce pure proteins for direct analysis of cellulase or other polysaccharide hydrolase activity. For some experiments, protein synthesis will be performed in the presence of wild-type and mutant scaffoldin genes, with the intent of generating novel cellulosomal structures. The resulting complexed and non-complexed proteins will be used in assays of cellulose hydrolysis, based on the cellodextrin-fermenting, cellulose-nondegrading bacterium Thermoanaerobacterium saccharolyticum B6A. Complexed and noncomplexed cellulases will be assayed in different combinations to test for enzyme synergy.