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D. Technical Highlights and Accomplishments

The activities and accomplishments of the MBGC project are deeply rooted in the collaborative environment that encompasses more than 10 research groups at EMSL/PNNL as well as 6 external research groups and involving nearly 30 scientists and students. In the following sections, detailed descriptions are provided of the progress to date and the future research and development plans for each of these groups. Much of the progress has also depended on the unique instrumentation and technologies at EMSL that are accessible to the project team. Large-scale systems biology projects such as this one are becomingly increasingly dependent on the use of expensive, one-of-a kind instruments that are not readily available at individual laboratories, or even many of the academic research institutions. The presence of (a) high-performance mass spectrometers, (b) NMR and EPR instruments, (c) innovative biological imaging instruments, and (d) large-scale computational facilities and expertise at EMSL provides an ideal environment for today’s large scale genomics and post-genomics based systems biology projects. The MBGC project, in many ways, provides a testament to this assertion.

Selected technical accomplishments of the MBGC team include:

Development of a new bioreactor design that provides a basis for uniform cultivation of microbial samples for the entire MBGC team. A standardized platform was developed that allows different teams at different locations to cultivate and grow cyanobacteria samples consistently and uniformly. This enables tailored experiments that can be done much more expediently, allows cross-laboratory comparisons of cultivation conditions, and provides for confirmation and validation of results and observations. Three standardized bioreactors were designed, built, and located at participant institutions (PNNL, Washington University, and Purdue University).
Proteome assisted genome annotation of Cyanothece that has resulted in the submission of a paper to Nature. PNNL/EMSL’s premier global proteomics capability, using accurate mass & time tag (AMT) technology, was used to ascertain and confirm existence of many previous hypothetical proteins, significantly improving the completeness and utility of the annotated genome. This is the first and most comprehensive effort of this combined genomics/proteomics approach, and will, we believe, set a new standard for genome annotation endeavors.
Determination of the first biomolecular structures of Cyanothece proteins and the delineation of protein structures for important nutrient transporters in cyanobacteria. Twelve protein structures have been determined by two research groups involved in the MBGC project. Several of these have been membrane-associated proteins responsible for delivering critical nutrients (Fe, Zn, HCO3-, and NO3-) to the cell.
Development of ScalaBlast tool for rapid homology studies at multiple genomes level. This tool, a very high-throughput sequence analysis engine, was used to perform high-speed BLAST calculations on the EMSL supercomputer. Over 2 CPU years of data analysis was performed. This capability will be made available to DOE’s Joint Genome Institute (JGI) as part of a new EMSL collaboration. This capability and the collaboration will become immediately useful to the MBGC project as the JGI is sequencing an additional 5-6 Cyanothece strains for comparative genomics study.
Development of a detailed picture of the ultrastructure of a cyanobacterial cell and its constituent biological membranes. Procedures have been developed to freeze Cyanothece cells so that inherent structural relationships of membranes, internal storage granules, and other organelles are preserved. New insights into photosynthetic membrane continuities and granule organization have resulted, using 3-D tomographic techniques to reconstruct entire volumes of the cell.
Network models of cyanobacterial circadian physiology based on global transcriptomics analysis. Two versions of transcriptome microarrays for Cyanothece have been designed and produced for the project. Gene expression profiles are used and modeled to better understand enzyme and metabolite pathways, and to deduce or infer the operative biology. Several intriguing insights regarding the diurnal cycle biochemistry of Cyanothece are resulting. The models developed provide a solid foundation for future research activities in this systems biology project.

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