U149259.pdf (15.52 MB)
Environmental gene screening
thesisposted on 2014-12-15, 10:33 authored by Helen C. Rees
DNA was directly extracted from a number of water and sediment samples taken from the soda lakes of the Kenyan-Tanzanian Rift Valley. A limited number of microbial specific enrichments of these were also extracted in the same way. 16S rDNA genes were amplified by the polymerase chain reaction (PCR) and microbial diversity studied using denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (t-RFLP). DGGE had relatively poor resolution, as shown by the limited number of bands on gels as compared to the minimal number of amplicons represented by t-RFLP profiles. Cloning and sequencing of some of the DGGE bands indicated that they usually contained mixed amplicons. Amplicon sequencing enabled the identification of possible members of the soda lake community. Several sequences had high identity to the 16S rDNA genes of organisms previously isolated from soda lakes, others were only distantly related. Amplicons recovered from enrichment cultures were similar to those recovered from environmental DNA. In general, enrichment cultures showed comparable diversity to environmental samples in terms of DGGE and t-RFLP analysis.;Genomic DNA libraries were also made from DNA recovered from an environmental soda lake sample, and three different enrichment cultures. DNA was extracted from the samples and digested with Sau 3AI. Fragments of between 2 and 10kbp were gel purified and ligated into ZAP express vector (Stratagene) for packaging into the XL1-Blue MRF' Escherichia coli host cells. pBK-CMV phagemids were excised from the libraries and screened by direct enzyme assays for enzyme activities. Two cellulase and three esterase/lipase activity clones were found. Sequence data indicated that one cellulase activity clone had highest amino acid identity to an endo-beta-1,4-glucanase gene from the glycoside hydrolase family 5. The second active cellulase clone was not fully sequenced.
Date of award2002-01-01
Awarding institutionUniversity of Leicester