Wilkinson, Dianna E; (2001) Accessing Genes from Environmental DNA Libraries. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The classical approach for isolating enzymes from environmental samples is to enrich, isolate and screen a variety of microorganisms for the desired enzyme activity. The majority of the physiological diversity is excluded with this approach because it is estimated that >99% of the microorganisms observed in the environment cannot be cultivated. An alternative to this cultivation-dependent approach is to clone DNA which has been extracted directly from microbial biomass present in water, soil and sediment. Using an appropriate host system, enzyme activity can subsequently be detected by screening for heterologous gene expression. Geothermal regions are sources of thermophilic microbial diversity. This study sought to investigate the methods for extracting and cloning DNA from geothermal sediments for the purpose of detecting thermostable enzyme activities. Methods for extracting and purifying DNA directly from soil and sediment were evaluated based on DNA yield, purity and quality. Purified environmental DNA was then used to evaluate cloning protocols based on cloning efficiency, recombination efficiency and total number of recombinants generated per ligation reaction. Subsequently, two environmental gene libraries were constructed using a TA-cloning method with DNA directly extracted from sediments that were collected from Iceland geothermal sites. The environmental library designated as ICE16 was derived from biomass present in sediment at -74°C, pH 7.4, while the DNA used to construct library ICE22 was derived from biomass present in sediment at -58°C, pH 4.3. These libraries were screened for thermostable amylase, lipase, protease and phosphatase activities using established assays in both microtitre plate and indicator agar-plate formats. One transformant possessing phosphatase activity at 60°C (Phos22) and two transformants showing phenotypic differences on starch agar plates at 50°C (5ICE16, 6ICE16) were recovered from the ICE22 and ICE16 DNA libraries, respectively. These clones were selected for further evaluation including sequencing and expression studies.

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