SBIR-STTR Award

Sequencing and analysis of bacterial genomes has become routine in the development of diagnostics, drug targets and improved production strains. Problems with this approach include: (1) unassignable, hypothetical ORFs, comprising 25-50 percent of an average genome; (2) lack of hierarchical organization of information; and (3) insufficient connections to experimental biology, both in verification of predictions and in high throughput methods. IG (Integrated Genomics) has already produced gapped genomes of Enterococcus faecium and Fusobacterium nucleatum. The grant applicants propose to address the problems identified above using those bacteria as model organisms. In addition, the bacteria themselves have obvious importance as human pathogens. In Phase I, the investigators will use primer-walking to complete these gapped genomes, leaving 0 to 10 gaps. They will assign new and corrected ORFs, and make improvements in functional assignments using proprietary WIT-Pro(TM) genomic analysis software suite. The investigators go on to outline their goals for Phase II and Phase III of the project, which are (1) to correlate hypothetical ORFs with "orphan functions" missing from metabolic pathways, (2) to clone and over express selected ORFs and determine their enzymatic function(s), and (3) in collaboration with a second company Sigma-Genosis, to design proprietary gene expression arrays for commercialization.

Thesaurus Terms:
Fusobacterium nucleatum, Streptococcus enterococcus group, bacterial genetics, functional genomics, open reading frame DNA primer, computer program /software, genome, nucleic acid sequence molecular cloning

In completing Phase I of this grant, Integrated Genomics has produced an almost complete genomic sequence of Fusobacterium nucleatum (ATCC25586), leaving only 15 gaps. We have also assigned functions to ORFs using our propriety ERGO IM genomic analysis software suite. In Phase II, we will use our proprietary algorithms to correlate hypothetical ORFs with "orphan functions" missing from reconstructed metabolic pathways. Since F. nucleatum is known to be highly variable, we will completely sequence an additional hospital-isolated strain. We will choose this isolate based on its genetic distance from our sequenced strain. Further characterization of strain variability will be accomplished by SSH (suppressive subtractive hybridization), which will allow us to rapidly compare many other strains to our completely characterized isolates. We will sequence the resulting differences. We will design and manufacture F. nucleatum whole-genome microarrays and use them to probe the microbe's physiological states, some of which will mimic the stresses experienced during human infection. This will potentially reveal protective mechanisms and sensitive protein targets. Based on the results of strain-to-strain differences, we will develop a diagnostic chip to detect and rapidly characterize pathogenic states of F. nucleatum. Finally, we will over-express and enzymologically characterize selected protein targets found by the proposed combination of computational biology and whole-genome expression studies. Phase III will involve the verification of potential drug targets revealed in this study and commercialization of the proprietary DNA arrays. PROPOSED COMMERCIAL APPLICATION: Commercialization of this work include potential vaccines against certain novel Fusobacterium nucleatum proteins, and an antibiotic targeted against a unique Fusobacterium nucleatum biosynthetic pathway.

Thesaurus Terms:
Fusobacterium nucleatum, bacterial genetics, functional genomics, genetic mapping, open reading frame DNA primer, computer program /software, computer simulation, genome, molecular biology information system, nucleic acid sequence molecular cloning, subtraction hybridization