Construction and phenotypic screening of mid-size insert marine microbial environmental genomic libraries

Functional screening of environmental genomic libraries permits the identification of clones expressing activities of interest without requiring prior knowledge of the genes responsible. In this study, protocols were optimized for the construction of mid-size DNA insert, inducible expression environmental genomic plasmid libraries for this purpose. A library with a mean insert size of 5.2 kilobases was constructed with environmental DNA isolated from surface ocean water collected at Hawaii Ocean Time-series station ALOHA in plasmid cloning vector pMCL200 under the inducible control of the PLAC promoter. To begin to evaluate the utility of such libraries for gene expression-based screens, this library was screened phenotypically for clones expressing genes that confer fluorescence or distinctive coloration on colonies of host Escherichia coli cells, and results were compared to those for a fosmid library constructed from the same marine microbial DNA sample. Ecologically relevant sequences were identified in both libraries, and each was observed to offer both advantages and disadvantages. Results of this study suggest that mid-size insert plasmid libraries under the control of inducible promoters can provide a useful and complementary approach for both functional screening and shotgun sequencing of environmental genomic libraries.

Department dc:contributor.department

Joint Program in Oceanography/Applied Ocean Science and Engineering

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2008

Author dc:creator

• Braff, Jennifer C

Advisor dc:contributor.advisor

• Edward DeLong.

• /Woods Hole Oceanographic Institution. Joint Program in Oceanography/Applied Ocean Science and Engineering.
• Civil and Environmental Engineering.
• Woods Hole Oceanographic Institution.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/43722

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/43722