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Rotavirus infection is one of the six leading causes of death among children under the age of five years. Globally it causes more than 215 000 deaths annually of which 65% occur in low- and/or middle-income countries. The two licenced live-attenuated vaccines (Rotarix™ and RotaTeq™) tend to have a lower efficacy in low- and/or middle-income countries. The lower efficacy of rotavirus live-attenuated vaccines could be due to maternal antibodies and oral polio vaccines interfering with rotavirus vaccine uptake. Rotavirus live-attenuated vaccines have been associated with reassortment with circulating genotype strains. As alternative, subunit vaccines such as viral proteins can be considered. Rotavirus VP6 protein is considered as a candidate for subunit vaccine development. Rotavirus VP6 antibodies are responsible for long lasting immunity and the antibodies against VP6 block the release of the viral mRNA. Previous studies showed rotavirus VP6 provide heterologous protection by a significant reduction of virus shedding in mice and gnotobiotic pigs. Various recombinant yeasts were engineered by a previous MSc student, Mr M.S. Makatsa, using a unique yeast expression vector (pKM177). The recombinant yeasts contained an open reading frame (ORF) encoding rotavirus VP6 for the RVA/Human wt/ZAF/GR10924/1999/G9P[6] strain. The ORF was codon optimised to favour expression in Arxula adeninivorans (AO), Kluyveromyces lactis (KO) and Pichia pastoris/Pichia angusta (PO). However, there was no expression of VP6 optimised for expression in A. adeninivorans and K. lactis due to an additional out-of-frame ATG in the promoter region of the expression vector. In this study, the additional ATG was successfully removed by site-directed mutagenesis and the Kozak sequence was optimised to produce modified delATG_pKM177_AOVP6 and delATG_pKM177_KOVP6 constructs. The modified delATG_pKM177_AOVP6, delATG_pKM177_KOVP6 as well as delATG_pKM177_POVP6, the modified plasmid containing the VP6 ORF co, National Research Foundation (NRF), PRF

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 yea, Afrikaans: In hierdie studie, basispaaropeenvolgings wat kodeer vir VP2 en VP6 rotavirus strukturele proteïene van rotavirus stam RVA/Human-wt/ZAF/GR10924/1999/G9P [6] is gebruik in die konstruksie van ‘n wye verskeidenheid gis uitdrukkingsvektore met rotavirus strukturele proteïene VP2 en VP6 oopleesrame. VP2 en VP6 basispaaropeenvolgings is kodon geoptimaliseer vir uitdrukking in gis stamme K. lactis, A. adeninivorans en P. pastoris/H. polymorpha. Wye verskeidenheid gis uitdrukkingsvektore wat geoptimaliseerde oopleesrame van VP6 of VP2 bevat, is gebou vir uitdrukking van enkel proteïene in verskillende gisstamme. Vektore wat beide VP6 en VP2 geoptimaliseerde oopleesrame bevat is gebou vir gelyktydige uitdrukking van proteïene in verskillende gisstamme en om die vorming van dubbellaag rotavirus-agtige partikels toe te laat. 'n Totaal van agt gisstamme naamlik: Kluyveromyces marxianus, Kluyveromyces lactis, Debaryomyces hansenii, Yarrowia lipolytica, Hansenula polymorpha, Pichia pastoris, Candida deformans en Arxula adeninivorans is gekies vir sifting. Saccharomyces cerevisiae is ingesluit as 'n positiewe kontrole aangesien trippellaag rotavirus-agtige partikels (tlRLPs) voorheen suksesvol in hierdie gis geproduseer is. Al nege gisstamme is suksesvol getransformeer met pKM177 vektore wat gis geoptimaliseerde kodon VP6 oopleesrame bevat. Daar was egter slegs ses gisstamme waarin positiewe VP6 ORF integrasie gevind is. Die oorblywende drie gisstamme het higromisien B integrasie getoon, wat daarop dui dat die vektore suksesvol geïntegreer het in hierdie gisstamme, maar dat VP6 oopleesraam verloor is. Integrasie van beide rotavirus VP2/6 oopleesrame was suksesvol vir al die gis stamme wat met die dubbele uitdrukkingsvektore getransformeer is. Rotavirus VP6 proteïenuitdrukking is suksesvol uitgevoer in bakteriële selle. Die positiewe kontrole het ʼn spesifieke reaksie getoon met poliklonale rotavirus teenliggaampies wat teen Nebraska calf diarrhoea rotavirus opgewek is, National Research Foundation (NRF), Poliomyelitis Research Foundation (PRF)

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 availab