Consistent with the stated objectives of this RFA, the goal of this technology core is to furnish a suite of biochemical tools, reagents and assays that will serve as the basis for generating experimental evidence for the "biochemical function(s) of hypothetical genes and unknown ORFs, and function(s) of noncoding RNAs" by "generating targeted measurements." While equally important, methods for profiling of proteins and small molecules are far more limited in breadth and scope than those for genes and nucleic acids. The reasons for this disparity are multiple, but, to a large degree, reflect key technological limitations associated with the increased complexity of protein structure function relationships and the atomic, rather than molecular, diversity of biochemical reactions. Notwithstanding, this core leverages key technological expertise in (i) high throughput protein biochemistry, often found only in industrial settings or laboratories focused primarily, if not exclusively, on structure, rather than activity, to enable the rapid and efficient production and characterization of proteins of unknown function;and (ii) high resolution mass spectrometry-based metabolomics to enable massively parallel and conceptually unbiased biochemical screens and/or assays for protein activity in vitro and within the native biochemical milieu of the cell. With published, and largely pioneering, expertise in each of these areas specifically applied to Mycobacterium tuberculosis (Mtb), we now propose to organize these activities into a formal technology core that will serve and inform the biochemical activities of each research project. * Activity 1: Clone, express and purify recombinant Mtb proteins of unknown function for in vitro biochemical characterization and assays. * Activity 2: Conduct in vitro assays to identify substrates and products of these putative enzymes. * Activity 3: Develop in vitro biochemical assays for putative enzymes. * Activity 4: Perform unbiased metabolomic screening for protein function
TB is the leading bacterial cause of deaths worldwide and a pathogen of pandemic proportion. However, like the case for most microbes, its most unique attributes, and potentially most selective targets, are encoded by genes of unknown function. The establishment of the metabolomic and biochemistry core proposed herein will help fill this gap in knowledge.
|Kery, Mary Beth; Feldman, Monica; Livny, Jonathan et al. (2014) TargetRNA2: identifying targets of small regulatory RNAs in bacteria. Nucleic Acids Res 42:W124-9|
|DeJesus, Michael A; Ioerger, Thomas R (2013) A Hidden Markov Model for identifying essential and growth-defect regions in bacterial genomes from transposon insertion sequencing data. BMC Bioinformatics 14:303|