Identification and characterisation of novel cellulolytic genes using metagenomics
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Date
2010
Authors
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Journal ISSN
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Publisher
University of the Western Cape
Abstract
Metagenomics has been successfully used to discover novel enzymes from uncultured microorganisms in the environment. In this study, metagenomic DNA from a Malawian hot spring soil sample was used to construct a fosmid library. This metagenomic library comprised of more than 10000 clones with an average insert size of 30 kb, representing more than 3.0 x 108 bp of metagenomic DNA (equivalent to approximately 100 bacterial genomes). The library was screened for cellulase activity using a Congo red plate assay to detect zones of carboxymethylcellulose hydrolysis. This yielded 15 positive fosmid clones, of which five were further characterised for activity and thermostability using the 3, 5-dinitrosalicylic assay. Two of the five fosmids (XP008C2 and XP026G5) were selected for DNA pyrosequencing. The full sequence of the XP008C2 (29800bp) fosmid insert is presented in this study and genes thereon were chosen for further study.
Description
Masters of Science
Keywords
Molecular cloning, Methodology, Genetic engineering, DNA, Synthesis