Re-engineering the ribosome for efficient selenoprotein synthesis

i Acknowledgements ii Table of contents iii Abbreviations vi Chapter 1 Introduction 1 1.1 A ribosomal renaissance 1 1.2 Selenocysteine the 'unconventional' amino acid 10 1.3 The human selenoproteome 15 1.4 Selenocysteine incorporation 19 1.5 Progress towards efficient recombinant expression 21 1.6 A potential role for the ribosome in selenocysteine incorporation 26 1.7 Purview of thesis 29 Chapter 2 Materials and Methods 30 2.1 Chemicals 30 2.2 Enzymes 33 2.3 Bacterial media, stock solutions and buffers 34 2.4 General molecular biology 38 2.5 DNA sequencing and analysis 41 2.6 Protein electrophoresis and analysis 41 2.7 Bacterial host strains 42 2.8 Stock plasmids 46 2.9 Western blotting 48 2.10 Library construction 49 2.11 Chloramphenicol selection 50 2.12 5-Fluorouracil selection 50 2.13 Chlorophenylalanine selection 51 2.14 Chloramphenicol resistance assay 52

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