Browsing by Author "Anderson, Dominique Elizabeth"

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    Gene Discovery in Antarctic Dry Valley Soils
    (University of the Western Cape, 2008) Anderson, Dominique Elizabeth; Cowan, D.A.; Dept. of Biotechnology; Faculty of Science
    The metagenomic approach to gene discovery circumvents conventional gene and gene product acquisition by exploiting the uncultured majority of microorganisms in the environment. It was demonstrated in this study that metagenomic methods are suitable for gene mining in extreme environments that harbor very high levels of unculturable microorganisms. DNA was extracted from Antarctic mineral soil samples taken from the Miers Valley, Antarctica. The metagenomic DNA was also used to construct a fosmid library comprising over 7900 clones with an average insert size of 29 kb. PCR amplification using bacterial and archaeal 16S rRNA gene specific primers and subsequent denaturing gradient gel electrophoresis (DGGE) of bacterial 16S rDNA amplicons showed that a small percentage of bacterial diversity was captured in the metagenomic fosmid library. Activity-based screening for lipase and esterase genes using a tributyrin plate assay yielded twelve positive clones. LD1, a putative, novel cold-active GDSL lipase/esterase was identified and sequenced. The C-terminal domain of the ORF was found to be an autotransporter similar to those associated with type V secretion systems in Gram negative bacteria. Sub-cloning of the gene resulted in lipolytic activity in E. coli. Preliminary enzyme assays have determined that LD1 hydrolyses p-nitrophenyl esters with chain lengths shorter than C10, an indication that the enzyme is an esterase. Complete purification and characterisation of this enzyme is subject to further study.
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    Medical biorepositories of South Africa: establishing a medical biorepository network in South Africa to advance health research
    (Biopreservation and Biobanking, 2025) Anderson, Dominique Elizabeth; Conradie, Engela Helena; Fransman, Warren Oswald
    The metagenomic approach to gene discovery circumvents conventional gene and gene product acquisition by exploiting the uncultured majority of microorganisms in the environment. It was demonstrated in this study that metagenomic methods are suitable for gene mining in extreme environments that harbor very high levels of unculturable microorganisms. DNA was extracted from Antarctic mineral soil samples taken from the Miers Valley, Antarctica. The metagenomic DNA was also used to construct a fosmid library comprising over 7900 clones with an average insert size of 29 kb. PCR amplification using bacterial and archaeal 16S rRNA gene specific primers and subsequent denaturing gradient gel electrophoresis (DGGE) of bacterial 16S rDNA amplicons showed that a small percentage of bacterial diversity was captured in the metagenomic fosmid library. Activity-based screening for lipase and esterase genes using a tributyrin plate assay yielded twelve positive clones. LD1, a putative, novel cold-active GDSL lipase/esterase was identified and sequenced. The C-terminal domain of the ORF was found to be an autotransporter similar to those associated with type V secretion systems in Gram negative bacteria. Sub-cloning of the gene resulted in lipolytic activity in E. coli. Preliminary enzyme assays have determined that LD1 hydrolyses p-nitrophenyl esters with chain lengths shorter than C10, an indication that the enzyme is an esterase. Complete purification and characterisation of this enzyme is subject to further study.
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    Metagenome sequencing and in silico gene discovery: From genetic potential to function
    (University of the Western Cape, 2012) Anderson, Dominique Elizabeth
    In a previous study, metagenomic DNA extracted from Antarctic Dry Valley soils was used to construct a large contig bacterial shotgun fosmid library (Anderson, 2008). In the current study, clones were selected based on a functional screen for putative lipolytic enzymes, which incorporated tributyrin in agar screening plates. Clones were subsequently subjected to next-generation sequencing and bioinformatic analysis, which allowed for further investigation of a portion of the Antarctic metagenome. Assembly and annotation of the genetic data encoded on three fosmid clones allowed for the identification of the genes responsible for tributyrin hydrolysis. Furthermore, hypotheses relating to survival and adaptation to abiotic conditions prevalent in the extreme Antarctic environment were developed (Chapter 3). A cold adapted esterase was subsequently characterised and showed substrate preference for para-nitrophenyl propionate. The optimum temperature and pH for the enzyme, DEaseI was 25 ° C and 8.5, respectively. In addition, results indicated that DEaseI was sensitive to thermal inactivation (Chapter 4). Furthermore, in fosmid clone LD13, one particular ORF annotated as a Water HYpersensitity response protein, became the focus of further study. When sub-cloned into a heterologous host, both ionic and osmotic stress tolerance was observed in vivo. The protein also exhibited a cryoprotective function in vitro, preventing cold denaturation of malate dehydrogenase during cycles of freeze-thaw (Chapter 5). This study demonstrates the value of combinatorial in silico and ‘-omic’ based techniques for the discovery and functional characterisation of potentially novel genes from bacteria which inhabit Antarctic Dry Valley soils.