Contents... vii. List of Figures... xii. List of Tables... xiv. Abbreviatons... xv. Summary... xvii. 1. Introduction In vitro evolution...

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1 vii Contents Contents... vii List of Figures... xii List of Tables... xiv Abbreviatons... xv Summary... xvii 1. Introduction In vitro evolution Phage Display Technology Cell surface display Bacterial display system Yeast surface display Other cell surface display Ribosome display mrna display CIS and CAD display In vitro compartmentalization Chemistry of SNAP-BG covalent interaction Aim of the Present Study Concept of multi-copy bead display approach Materials... 28

2 viii 2.1 Bacterial Strains Nucleic acids Plasmids Oligonucleotide Oligonucleotides for multi- copy Bead display Other nucleic acids Antibodies Enzymes Restriction endonuclease Other Enzymes Chemicals and reagents Special chemicals and reagents for Multi-copy bead display reagents Kit reagents Media, Buffers and solutions Media for bacteria culture Preparation of Buffers and solution for nucleic acid biochemistry Buffers and solution for Multicopy Bead Display Standards DNA size Standards Protein molecular weight marker Device instruments Device for Emulsion Preparation Methods Purification of DNA by ethanol precipitation Analytical isolation of plasmid DNA Preparative isolation of plasmid DNA Preparative isolation of plasmid DNA from agarose gel... 41

3 ix Determination of DNA concentration Digestion of plasmids with restriction endonucleases Dephosphorylation of 5 ends of DNA Separation of DNA by agarose gel electrophoresis DNA ligation Transformation of chemically competent bacteria Polymerase chain reaction (PCR) Analysis of proteins Construction and characterization of model templates Western Blot analysis of GFP-SNAP fusion protein BG-Binding assay of GFP-SNAP Covalent coupling of oligonucleotides to microbeads Verification of primer coupling on microbeads through probe hybridization Covalent coupling of oligonucleotide to BG Water in oil emulsion PCR Determination of size distribution of picoliter reactors SYBR green staining of PCR products on beads Probe hybridization to PCR products on microbeads Normalization of BG binding sites on beads with BG-oligo Cell free expression of proteins in water in oil emulsion Antibody staining of microbeads Analysis of beads through Flow Cytometry Generation of beads containing GFP-SNAP and MS2-SNAP through competition Construction of a T7 promoter library Screening of beads displaying the T7 promoter library by flow cytometry Re-amplification of DNA from beads Generation of expression cassettes by overlapping PCR... 56

4 x 3.15 Sequencing of T7 promoter variants Cloning of T7 promoter variants IntroducingT7 promoter variants upstream of luciferase gene Expression of T7 promoter variants in luciferase assay system Production of RNA for transcription assay Real time PCR analysis of RNA transcripts Production of RNA for translation assay Translation assay through luciferase assay Results Templates used for the development of Multi-copy Beads Display Creation of stable emulsions Analysis of picoliter reactors PCR in water in oil emulsion Amplification of gene products through empcr PCR on microbeads in emulsion Standardization of PCR on beads in water in oil emulsion Expression of protein in water in oil emulsion Construction and expression of Proteins Expression of fusion protein in IVTT in emulsion Coupling of DNA and encoded proteins to beads by emulsion PCR and emulsion IVTT Normalization of BG binding sites with BG oligo hybridization Flow cytometric analysis of Protein on beads Clonality and sensitivity of the multi-copy bead display approach Estimation of number of DNA molecules per bead in multi-copy bead display Application of Multi-copy Bead Display Approach T7 promoter library and its display Bead display and selection of T7 promoter variants... 85

5 xi 4.9 Characterization of the selected T7 promoter variants Optimization of a luciferase assay system for analyzing promoter variants Quantitative characterization of T7 promoter variants through luciferase assay Sequence analysis of promoter variants Characterization of the C62 T7 promoter variant Comparison of protein expression kinetics of C62 and wildtype T7 promoter Effect of promoter variation on transcription and translation Mutation studies on the new T7 promoter variant C Discussion Multi-copy bead display system In vitro evolution of a novel T7 promoter variant Scope of Multicopy Bead Display Approach Bibliography Appendix Patents Publications Conference contributions Curriculum vitae