Single Cell Genomics of Hyperthermophiles

70588 Modern methods of molecular analysis reveal an astonishing discrepancy between the diversity and numbers of organisms present in natural habitats and the relatively few capable of being cultured. Single Cell Genomics is a new initiative to develop the tools and methods for extracting cultivation-resistant genetic diversity in a form that is useful for whole genome sequence analysis and expression screening. This project will apply these tools to discover valuable hyperthermophilic enzymes and sequence information from a class of microorganisms especially difficult to grow in the lab. In Phase I, single microorganisms will be isolated from a diverse mixture using microscopy and a micromanipulator. Specific tasks include the isolation of a single molecule of intact genomic DNA, unbiased amplification with high fidelity, ligation to a new dual orientation zero-background cloning vector, and transformation into a new E. coli strain. A random genomic library from a single microbial cell adequate for 10X sequence coverage will be constructed. The long-term goals of this research are to develop a phylogenetically diverse set of genomic libraries from single cells of culture-resistant hyperthermophiles and screen them for commercially valuable enzymes. Commercial Applications and Other Benefits as described by the awardee: The technology could result in sales of reagent kits, a single cell genomic DNA cloning service, and hyperthermophilic genomic libraries. Enzyme activity screens for hyperthermophilic laccase and xylanase activities should provide environmentally friendly new reagents for the fiber processing and wood pulping industries.