Masters Degrees (Microbial, Biochemical and Food Biotechnology)

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Browsing Masters Degrees (Microbial, Biochemical and Food Biotechnology) by Author "Albertyn, Jacobus"

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    Engineering yeast strains for the expression of South African G9P[6] rotavirus VP2 and VP6 structural proteins
    (University of the Free State, 2015-02) Makatsa, Mohau Steven; O’Neill, Hester G.; Albertyn, Jacobus
    English: In this study, sequences encoding VP2 and VP6 rotavirus structural proteins from rotavirus strain RVA/Human-wt/ZAF/GR10924/1999/G9P[6] were used in construction of wide-range yeast expression vectors containing the ORFs encoding rotavirus structural proteins VP2 and VP6. VP2 and VP6 sequences were codon-optimized for expression in yeast strains K. lactis, A. adeninivorans and Pichia pastoris/Hansenula polymorpha. Wide-range yeast expression vectors containing either VP6 or VP2 yeast optimized ORFs were constructed for expression of single proteins in different yeast strains. Dual vectors containing both VP6 and VP2 yeast optimized ORFs were constructed to allow simultaneous expression of proteins in different yeast strains and to enable the formation of double-layered rotavirus-like particles. A total of eight yeast strains namely: Kluyveromyces marxianus, Kluyveromyces lactis, Debaryomyces hansenii, Yarrowia lipolytica, Hansenula polymorpha, Pichia pastoris Candida deformans and Arxula adeninivorans were selected for screening. Saccharomyces cerevisiae was included as a positive control as triple-layered rotavirus virus-like particles (tlRLPs) have been successfully produced in this yeast before. All nine yeast strains were successfully transformed with pKM177 vectors containing yeast codon-optimized VP6 ORFs. However, only six yeast strains indicated positive VP6 ORF integration. The other three yeast strains indicated no VP6 integration but hygromycin B integration suggesting that the vectors successfully integrated in these yeast strains but VP6 ORF was lost. Integration of both rotavirus VP2/6 ORFs was evident in all yeast strains transformed with the dual expression vectors. A control to monitor rotavirus VP6 protein expression in yeast was successfully prepared in bacterial cells. The positive control indicated specific reaction when polyclonal rotavirus antibody raised against Nebraska calf diarrhoea rotavirus strain. Rotavirus VP6 expression in yeast strains shown no reactions for K. lactis and A. adeninivorans codon-optimized VP6 ORF in all six yeast strains that tested positive for intergration of the VP6 ORF. All yeast strains except K. marxianus showed possible reaction for VP6 expression for P. pastoris/H. polymorpha codon-optimized VP6 ORF although the identity of VP6 should be confirmed.
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    Exploring carbon cycling in selected micro-organisms exposed to terrestrial carbon sequestration
    (University of the Free State, 2014-02) Chen, Jou-an; Van Heerden, Esta; Albertyn, Jacobus; Erasmus, Mariana
    South Africa‘s economy is primarily driven by the utilization of coal to provide electricity, which results in more fossil fuels to be burnt that contributes towards global warming. The average daily temperature is estimated to rise between 1.1 to 6.4˚C by 2100. Carbon sequestration is a technology that can limit CO2 emission into the atmosphere by storing the CO2 away in oceans or the terrestrial subsurface. South Africa is focusing on geological storage at depths of 1 000 m. Limited scientific knowledge is available on the direct impact when large amounts of supercritical CO2 is injected into the subsurface. This includes the diversity of the deep subsurface microbial communities as well as their ecosystems and biogeochemical processes. The main aim of this project was to use selected deep subsurface micro-organisms (T. scotoductus, Geobacillus sp. GE-7 and Geobacillus sp. A12) and an organism that was known to grow under pressure (E. limosum) and introduce them to CCS conditions using a high pressure syringe incubator system. The identities of the selected micro-organisms were verified using molecular techniques, the genomes of these micro-organisms were retrieved and information regarding possible CO2 fixation pathways was verified using the Metacyc database collection. The CO2 fixation pathways of interest were the Calvin cycle, the reductive acetyl Co-enzyme A and the reductive citric acid cycles. Surprisingly, T. scotoductus and E. limosum were able to remain viable and metabolically active even at 100 bar and 100% CO2. This has never been previously reported in literature. However Geobacillus sp. GE-7 and Geobacillus sp. A12 could not remain viable when the pressure was increased from 2 bar to 20 bar or higher. The outcomes of this study indicate that the interactions between supercritical CO2 and the subsurface organisms should be considered as biogeochemical cycling. However, these interactions in the subsurface are still relatively unknown and the availability of interactive metabolic pathways indicate that the subsurface communities could survive and interact with this introduced substrate.