M. Tech. (Department of Biotechnology, Faculty of Applied and Computer
Sciences), Vaal University of Technology. / The use of conventional culture-based approach results in vast majority of microbes (90 - 99%)
unaccounted for. However, over the past years, the use of metagenomics, which is a culture-independent
comprehensive approach has enabled researchers to access nearly 100% of the microbiome. In this
study, three hot springs (44 – 70 oC) in Limpopo province of South Africa were investigated as potential
sources of genes encoding for DNA-manipulating enzymes (DNA polymerase, DNA ligase and
endonuclease), which are central in genetic engineering. They are usually grouped into four broad
classes (nucleases, ligases, polymerases and modifying enzymes) depending on the type of the reaction
they catalyze. Accordingly, hot spring metagenomic DNA was successfully extracted using modified
SDS-CTAB method involving gel purification and electroelution. Consequently, a portion of the
extracted metagenomic DNA was used for sequencing and another for fosmid library construction.
Sequencing was done using Illumina MiSeq next generation sequencing platform and sequence data
analyzed and de novo-assembled using CLC Genomic Workbench, which resulted in 5 681 662 reads
and 7 338 contigs. A metagenome expression fosmid library of approximately 2.16 x 103 clones was
also constructed using CopyControl™ HTP Fosmid Library Production Kit with pCC2FOS™ Vector.
A BLAST algorithm in NCBI revealed 57 distinct genes for DNA polymerase, 29 genes for DNA ligase
and more than 100 genes for endonuclease II enzymes. Hence, three genes related to thermophiles
representing genes for DNA polymerase, DNA ligase and endonuclease II were selected. Accordingly,
the three genes were codon-optimized, synthesized and successfully cloned into pET- 30a (+) and
overexpressed in Escherichia coli BL21 (DE3) by inducing with 0.5 mM IPTG and incubating
overnight at 16ºC. The cells were lysed using B-PER Reagent, protein extracted and purified using
AKTA start protein purification system and purity of 85- 95 % was achieved. From this study, it can be
concluded that metagenomics as an approach, can be used to mine for putative DNA-manipulating
enzymes from hot spring metagenome. Besides, further study should be conducted to formulate the
developed DNA-manipulating enzymes and study the practical application and chart way for
commercialization. Moreover, the constructed fosmid library could also be screened for potentially
novel thermo-stable biomolecules of industrial and therapeutic importance.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:vut/oai:digiresearch.vut.ac.za:10352/488 |
| Date | 09 1900 |
| Creators | Mokoena, Morena India |
| Contributors | Feto, Naser Aliye, Dr, Rashamuse, K., Dr. |
| Publisher | Vaal University of Technology |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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