Single Cell Genomics of Hyperthermophiles

70588S02-II Less than 1% of the microbes in the environment can be cultured in the lab. Therefore, to reveal the full genetic diversity of microbial communities, this project will develop technology to clone the entire genome of a single microbial cell. The approach will allow whole genome sequencing and expression screening of any cell, particularly those that exist in extreme environments. The approach will be applied to reveal the genomic diversity of hyperthermal environments and to discover valuable heat-stable enzymes encoded by individual cells in these communities. Genomic libraries and enzyme activities will be developed for academic and commercial use. In Phase I, single microorganisms were isolated from a mixture of cells, using fluorescent microscopy and a micromanipulator. The genome of a single cell was amplified and sheared to 1-2 kb fragments. It was then ligated to a transcription-free, zero-background cloning vector and transformed into a high efficiency E. coli strain. A random shotgun library, adequate for 10X sequence coverage, was constructed from this cell's genome to demonstrate the feasibility of single-cell genomics. In Phase II, genomic libraries of 70 novel hyperthermophilic microbes will be constructed. All 70 libraries will be screened extensively by plate-based colony expression assays for enzymes with commercial and academic interest. Enzymes with the greatest potential value will be developed and commercialized. Commercial Applications and Other Benefits as described by awardee: The technology should generate a single-cell DNA amplification kit, dual expression vector, and highly competent E. coli cells to be sold as reagent kits. A single-cell genomic DNA cloning service would be offered. Hyperthermophile genomic libraries could be sold as a series of discovery products. Enzyme activity screens for numerous hyperthermophilic activities could provide environmentally friendly new reagents for numerous industries. Potential applications also may exist in other fields, such as microbial pathology and environmental genomics, in which numerous microorganisms are known to exist but cannot be cultured.