Genomes to Life Contractor-Grantee Workshop III
February 6-9, 2005, Washington, D.C.
Environmental Genomics
66
Application of High Throughput Microcapsule Culturing to Develop a Novel Genomics Technology Platform
Martin Keller1* (mkeller@diversa.com), Karsten Zengler1, Marion Walcher1, Carl Abulencia1, Denise Wyborski1, Sherman Chang1, Imke Haller1, Trevin Holland1, Fred Brockman2, Cheryl Kuske3, and Susan Barns3
1Diversa Corporation, San Diego, CA; 2Pacific Northwest National Laboratory, Richland, WA; and 3Los Alamos National Laboratory, Los Alamos, NM
Project Description
The overall goal of this project is to demonstrate the combination of high-throughput cultivation in microcapsules, which gives access to previously uncultivated microorganisms, with genome sequencing from one to a few microcolony-containing microcapsules. This will allow direct access to physiological and genomic information from uncultured and/or difficult-to-culture microorganisms. This approach is fundamentally different than characterization and/or assembly of shotgun or BAC clones derived from community DNA or RNA. The units of analysis in our approach are living, pure microbial cultures in microcapsules, as opposed to the disassembled mixture of small fragments of genomes and cellular networks that have lost their biological context in studies using community nucleic acids. It is envisioned that the microcapsule based, high-throughput cultivation method will also be combined with Transcriptomics and Proteomics technology in the future.
Project achievements
A high-throughput cultivation method based on single cell encapsulation in microcapsules in combination with flow cytometry has been applied to two soil samples. It has been demonstrated that microorganisms belonging to a variety of bacterial phyla, such as Acidobacteria, Gemmatimonadetes and candidate division TM7, were growing in the microcapsules and subsequently formed microcolonies.
A fluorescent in situ hybridization (FISH) method has been optimized to selectively target and sort encapsulated microcolonies of interest using fluorescence activated cell sorting.
A whole-genome amplification technique has been employed to acquire a sufficient mass of DNA from targeted, encapsulated microcolonies (E. coli) to generate libraries for shotgun sequencing of entire genomes. The whole-genome amplification has been optimized so that DNA from as few as two cells can be amplified routinely. The genome coverage of amplified FISH-targeted microcolonies was evaluated using microarrays. Microarray data demonstrated a genome-coverage of 97% to 99% without high levels of bias towards certain genes. Subsequently, genomic libraries have been constructed from these amplified DNA samples. Almost 300 clones of each library have been sequenced. Sequence analysis confirmed that 70% of the clones originated from E. coli DNA. Additional gene sequences were affiliated with certain beta-Proteobacteria typically found as experimental contaminants.
This work was supported by the Office of Science (BER), U.S. Department of Energy, Grant No. DE-FG02-04ER63771.
* Presenting author
