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Isolation of dhlA Gene Responsible for Degradation of 1, 2-dichloroethane from Metagenomic Library Derived from Daecheong Reservoir  

Kang, Cheol-Hee (Department of Microbiology, Chungbuk National University)
Moon, Mi-Sook (Department of Microbiology, Chungbuk National University)
Song, Ji-Sook (Department of Microbiology, Chungbuk National University)
Lee, Sang-Mhan (Department of Life Science, Chongju University)
Kim, Chi-Kyung (Department of Microbiology, Chungbuk National University)
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Abstract
Traditional screening techniques have missed up to 99% of microbial resources existing in the nature. Strategies of direct cloning of environmental DNAs comprising tine genetic blueprints of entire microbial metagenomes provide vastly more genetic information than is contained in the culturable. Therefore, one way to screening the useful gene in a variety of environments is the construction of metagenomic DNA library. In this study, the water samples were collected from Daecheong Reservoir in the mid Korea, and analyzed by T-RFLP to examine the diversity of the microbial communities. The crude DNAs were extracted by SDS-based freezing-thawing method and then further purified using an $UltraClean^{TM}kit$ (MoBio, USA). The metagenomic libraries were constructed with the DNAs partially digested with EcoRI, BamHI, and SacII in Escherichia coli DH10B using the pBACe3.6 vector. About 14.0 Mb of metagenomic libraries were obtained with average inserts 13 ${\sim}$ 15 kb in size. The genes responsible for degradation of 1, 2-dichloroethane (1, 2-DCE) via hydrolytic dehalogenation were identified from the metagenomic libraries by colony hybridization. The 1, 2-dichloroethane dehalogenase gene (dhlA) was cloned and its nucleotide sequence was analyzed. The activity of the 1, 2-DCE dehalogenase was highly expressed to the substrate. These results indicated that the dhlA gene identified from the metagenomes derived from Deacheong Reservoir might be useful to develop a potent strain for degradation of 1, 2-DCE.
Keywords
metagenomic libraries; dhlA; 1, 2-dichloroethane dehalogenase; T-RFLP;
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