Identification of the genes encoding enzymes involved in the synthesis of the biopolymer paramylon from Euglena gracilis

iii Opsomming iv Acknowledgements v List of Figures ix List of Tables x Abbreviations xi Review of Literature 1 1.1 Euglena gracilis 1 1.2 Taxonomy 2 1.3 Paramylon and paramylon synthesis 3 1.4 Glycosyl transferases 6 1.5 Partial purification of β-1,3-glucan synthases and identification of catalytic subunits 8 1.6 Paramylon synthase 11 1.7 Cytokine–related immunopotentiating activities of paramylon 11 1.8 Aims and experimental approach 13 Materials 15 2.1 Chemicals and kits 15 2.2 Primers 15 2.3 Primers designed from an isolated E. gracilis β-glucan synthase sequence 17 2.4 Web – based programs 17 Methods 18 3.1.1 Construction and screening of the cDNA library 18 3.1.1.1 Growth conditions of Euglena gracilis 18 3.1.1.2 Total RNA extraction 18 3.1.1.3 Isolation and quantification of mRNA 18 3.1.1.4 cDNA synthesis 19 3.1.1.5 cDNA library construction and in vitro packaging of the phage library 19 3.1.1.6 Amplification of the cDNA primary library 19 3.1.1.7 Mass excision of the amplified library 19 3.1.1.8 Single clone excision 20 3.1.2 Screening of the phage library 20