Project Description
| With over 600 genomes with complete sequences currently available in public databases and thousands of genome sequencing projects underway, there’s a pressing need to effectively annotate genomic sequences quickly and accurately for functional activity in a high throughput manner. The main objective of this project is to experimentally annotate (assign a biochemical function) a large group of conserved hypothetical proteins using high throughput protein production and enzymatic screening methods. This approach for experimental annotation will be applied to hypothetical proteins from a prokaryote and a eukaryote of programmatic interest. In the first stage of the project we will functionally map the set of conserved hypothetical proteins from Shewanella oneidensis which contains ~800 members. The rationale for screening the set of conserved hypothetical from a single genome is to benchmark the utility of the enzymatic screening approach for the improvement of functional assignment for a large set of proteins of unknown function. A second component of the project will be to apply a directed screening strategy to uncharacterized proteins of the haloacid dehalogenase (HAD)-like hydrolase superfamily, which will be tested for the presence of dehalogenase, phosphonatase, phosphatase, or glucomutase activities for HAD-like hydrolases. This aim will use several strains of Shewanella and the symbiotic fungus Laccaria bicolor. Targets from L. bicolor will be amplified from cDNA clones, clone libraries or generated using a PCR-based gene synthesis approach. This objective will provide a foundation to assess the capabilities for specific functional assignments for a substantial number of unknown prokaryotic and eukaryotic proteins. For protein production, we will use the efficiency of automated strategy to implement a parallel pipeline consisting of an E. coli and yeast expression systems. The screening strategy uses a tiered approach where targets are categorized using a series of general screens and then rescreened for specific functional assignments using a directed series of natural substrates. The general screening assays have relaxed substrate specificity and are designed to identify the subclass or sub-subclasses of enzymes (phosphatase, phosphodiesterase/nuclease, protease, esterase, dehydrogenase, and oxidase) to which the unknown protein belongs. Further biochemical characterization of proteins can be facilitated by the application of secondary screens with natural substrates (substrate profiling). |
Conserved Hypothetical Proteins from Shewanella Oneidensis MR-1
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Project Pipeline
