Construction of environmental DNA library and specific detection of virulent Vibrio vulnificus from sediments of tidal flat

학위논문(박사)--서울대학교 대학원 :생명과학부,2005.Docto

Detection of Vibrio vulnificus in marine samples

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Discovery of a novel lipase from the environmental DNA library

A metagenomic library was constructed using a fosmid vector from deep sea sediment in clam beds area occupied by low-temperature vents on Edison seamount (depth, 1440m), western Pacific. The total DNA was extracted from the sediment sample by grinding-method and high molecular weight environmental DNA (>30kb) was directly eluted from the gel containing PVP (polyvinylpyrrolidone). Cloning of the environmental DNA into a fosmid vector has been conducted successfully and the library analysis revealed that it contained 8,823 clones with average size of 32.3 kb, and the total size was equivalent to 284 Mb. From the screening of activities, one positive clone for lipase, one for protease and 31 for β-lactamase were found. One lipase clone (ES01029D12) has been chosen for further analysis. The insert DNA (36.7kb) was completely sequenced. The 26 open reading frames (ORFs) was obtained by using ORF Finder, most of them were related to the protein of gamma proteobacteria. We found the two lipase-related ORFs, hypothetical lipase (915 bp) and lysophospholipase (600 bp), which showed 28 and 35% identities, respectively. Both predicted lipases contained a different active-site consensus region, GHSKG and GHSMG. The former was subcloned and its lipolytic activity was confirmed. The metagenomic study showed that the previously undiscovered microbial diversity could be found from the environmental metagenomes and this approach could be applicable to look for novel biocatalysts.1

Construction of metagenomic libraries from Edison seamount sediments and screening of metabolic activities

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