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

Abstract

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.