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dc.contributor.advisorSimmonds, Robin
dc.contributor.authorNones, Christopher-Oliver Mortera
dc.date.available2015-05-26T03:57:59Z
dc.date.copyright2015
dc.identifier.citationNones, C.-O. M. (2015). Cloning and Characterization of Diacylglycerol Acyltransferase in Escherichia coli and Rhodococcus opacus PD630 (Thesis, Master of Science). University of Otago. Retrieved from http://hdl.handle.net/10523/5676en
dc.identifier.urihttp://hdl.handle.net/10523/5676
dc.description.abstractBiodiesels are increasing in popularity as prices of petroleum have increased over the past two decades and triacylglycerols (TAG) are an attractive alternative as a feedstock to produce biodiesel. Rhodococcus opacus PD630 is an environmental organism well characterized for its ability to accumulate TAG to a high percentage of its cell dry weight. It is known that the accumulation of TAG by R. opacus PD630 is dependent on a high carbon to nitrogen ratio. To improve the attractiveness of R. opacus PD630 as a source of TAG, cheaper waste substrates such as food wastes must be utilized, however, these substrates are likely to be inhibitory to TAG accumulation. Glycerol is a cheap low nitrogen feedstock derived from the production of biodiesel from TAG. Growth of R. opacus PD630 on glycerol as a sole carbon source was examined and the bacterium was unable to grow in the broth culture. Diacylglcyerol acyltransferase (DGAT) is the rate-limiting enzyme in the accumulation of Triacylglcyerols. If TAG accumulation could be controlled independently of host regulation, a wide range of waste substrates could be utilized. To address this, a recombinant E. coli, containing vector pDGAT harboring an IPTG inducible His-tagged DGAT gene, was characterized for its protein expression and lipid accumulation. An optimized electroporation protocol was developed to clone pDGAT into R. opacus PD630 and the expression of DGAT examined. E. coli DGAT was able to produce DGAT protein when induced with IPTG while R. opacus DGAT A, an erythromycin resistant transformant produced DGAT constitutively. A second erythromycin resistant transformant was unable to produce DGAT and was shown to not harbor pDGAT. Sequencing of pDGAT revealed it was never successfully constructed and that this plasmid was in fact pQE-80L-DGAT.
dc.format.mimetypeapplication/pdf
dc.language.isoen
dc.publisherUniversity of Otago
dc.rightsAll items in OUR Archive are provided for private study and research purposes and are protected by copyright with all rights reserved unless otherwise indicated.
dc.subjectBiodiesel
dc.subjectE.coli
dc.subjectRhodoccous
dc.subjectopacus
dc.subjectTAG
dc.subjectTriacylglycerol
dc.subjectDGAT
dc.subjectDiacylglycerol Acyltransferase
dc.subjecttransformation
dc.titleCloning and Characterization of Diacylglycerol Acyltransferase in Escherichia coli and Rhodococcus opacus PD630
dc.typeThesis
dc.date.updated2015-05-26T03:21:49Z
dc.language.rfc3066en
thesis.degree.disciplineMicrobiology and Immunology
thesis.degree.nameMaster of Science
thesis.degree.grantorUniversity of Otago
thesis.degree.levelMasters
otago.openaccessOpen
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