Therapeutic recombinant proteins such as antibodies, hormones, enzymes, and anticoagulants can be used in medicine, for food production, agriculture and bioengineering industries. Recombinant proteins are currently being produced by mammalian, plant, insect and microbial culture systems but microalgae, when used as an expression vector, would offer many benefits over existing methods. These advantages include, lower production costs, faster growth rates, easier culturing, simpler transgenic manipulation as well as modified abilities of transcription and translation. South Africa boasts one of the most bio-diverse regions globally, with tremendous species richness in freshwater eco-systems with the potential of commercially desired, novel microalgae species.
To investigate the feasibility of establishing microalgal biotechnology in South Africa, the model green microalgae species, Chlamydomonas reinhardtii was transformed under available conditions. Furthermore, five hundred microalgal isolates previously collected across South Africa and recorded as part of the Microalgae Culture Collection held at the Council for Scientific and Industrial Research (CSIR), were bio-prospected for their growth potential.
First a gene encoding the Chlamydomonas codon optimised green fluorescent protein (CrGFP) was cloned into a pChlamy4 vector under the control of the chimeric Hsp70A-rbcS2 promoter and selected for with a bleomycin resistant gene, she ble. The verified plasmid was then electro-transformed into the nuclear region of two C. reinhardtii strains CC-125 and CC-400. The microalgae transformants were assessed at the DNA level by PCR and further at the CrGFP protein production level by fluorescence spectrophotometry.
From the Microalgae Culture Collection Database, forty isolates were chosen microscopically based on whether they are unicellular, non-colonial, with large or many chloroplasts and a thin cell wall. The isolates underwent a growth curve analysis and those with the best growth rates were then directly compared to C. reinhardtii spp. under phototrophic and mixotrophic growth conditions. These isolates were then identified by Sanger sequencing of the 18S rRNA region.
Results indicated successful insertion of the Crgfp transgene with detectable levels of CrGFP fluorescence above autofluorescence for all transformed C. reinhardtii colonies. In order of the greatest growth potential, microalgae with high growth rates were identified as C. sorokiniana, Chlorella sp., C. vulgaris, T. obliquus, T. dimorphus and R. subcapitata species. In this study, the South African Chlorella spp. isolates had a greater growth rate in both phototrophic and mixotrophic medium than the C. reinhardtii controls. In this study a foundation was laid for microalgae biotechnology research at the CSIR in South Africa, with C. reinhardtii as the reference organism; as well as considering the potential nuclear transformation in select indigenous species, such as Chlorella sp. for future microalgae protein expression systems. / Dissertation (MSc)--University of Pretoria, 2019. / NRF DST Innovation and Priority Research Areas Masters Scholarship / Plant Production and Soil Science / MSc / Unrestricted
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:up/oai:repository.up.ac.za:2263/71149 |
| Date | January 2019 |
| Creators | Stapelberg, Julanie |
| Contributors | Crampton, Bridget Genevieve, julanie.stapelberg@fabi.up.ac.za, Crampton, Michael C., Roth, Robyn |
| Publisher | University of Pretoria |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Dissertation |
| Rights | © 2019 University of Pretoria. All rights reserved. The copyright in this work vests in the University of Pretoria. No part of this work may be reproduced or transmitted in any form or by any means, without the prior written permission of the University of Pretoria. |
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