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Single-Cell Genomics of
Hyperthermophiles--Lucigen Corp.,
Dr. David A. Mead, Principal
Investigator,
Dr. David A. Mead, Business
Official,
DOE Grant No.
DE-FG02-02ER83484
Amount:
$750,000
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.