In the present work a modified version of yellow fluorescent protein containing an unnatural structural homologue of the natural amino acid pyrrolysine with a norbornene moiety was produced by expression in Escherichia coli. The incorporation of the unnatural amino acid was achieved by amber stop codon suppression method. A bio-othogonal click reaction was performed, binding a synthetic fluorescent dye to the modified protein. All steps towards necessary for obtaining the genetically modified organism were performed and documented. The artificial amino acid, as well as the dye used in the click reaction were synthetically prepared. The success of the project was demonstrated by LC/MS studies of the products. Fluorescence spectroscopy of click reaction product and the protein was performed, but no conclusive proof of FRET effects could as yet be made. This point remains of interest for future studies.
Table of contents
Abstract
Glossary
1 Introduction: Oligonucleotide Synthesis
2 Objective
3 Procedures and Materials
3.1 Chemistry
3.1.1 Methods and Materials utilized
3.1.2 Syntheses
3.2 Biology
3.2.1 Methods and Materials utilized
3.2.2 Primer Synthesis
3.2.3 Plasmid Mini Prep
3.2.4 Agarose Gel Electrophoresis
3.2.5 Polymerase Chain Reaction
3.2.6 Ligation
3.2.7 Transformation
3.2.8 Colony PCR
3.2.9 Protein Expression
3.2.10 Protein Purification
3.2.11 SDS-PAGE
3.2.12 Protein Digest
3.2.13 Protein Click Reaction
4 Results and Discussion
4.1 Agarose Gel Electrophoresis
4.2 SDS PAGE
4.3 Fluorescence Spectroscopy
4.4 Mass Spectrometry
4.4.1 MALDI-TOF
4.4.2 LC / MS
5 References
- Quote paper
- Anonymous,, 2014, Unnatural Amino Acid Incorporation and Click Chemistry, Munich, GRIN Verlag, https://www.hausarbeiten.de/document/416795
