The function of the halophilic dodecin [Elektronische Ressource] / Martin Grininger

Martin Grininger

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2006
• Nombre de lectures: 31
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades der Fakultät für
Chemie und Pharmazie der Ludwig-Maximilians-Universität München



The Function of the Halophilic Dodecin






Martin Grininger
aus Linz









2006


Erklärung

Diese Dissertation wurde im Sinne von §13 Abs. 3 bzw. 4 der Promotionsordnung vom 29.
Januar 1998 von Herrn Prof. Dr. Dieter Oesterhelt betreut.



Ehrenwörtliche Versicherung

Diese Dissertation wurde selbstständig, ohne unerlaubte Hilfe erarbeitet.




München, am 27. Juni 2006
…………………………….
Martin Grininger





Dissertation eingereicht am:

1. Gutachter: Prof. Dr. Dieter Oesterhelt
2. Gutachter: Prof. Dr. Karl-Peter Hopfner


Mündliche Prüfung am: 25. Oktober 2006













Meinen Eltern und Geschwister















Table of Contents

1. Introduction..................................................................................................................................................... 4
1.1 Archaea and Extremophiles ................................................................................................................. 4
1.2 Principles of halophilicity .................................................................................................................... 5
1.3 Cofactors Broaden the Spectrum of Protein Functions ........................................................................ 7
1.4 The Chemistry of Flavins..................................................................................................................... 8
1.5 Biosynthesis of Flavins ........................................................................................................................ 9
1.6 Flavin Degradation............................................................................................................. 11
1.7 Flavoproteins...................................................................................................................................... 12
1.8 Dodecin.............................................................................................................................................. 14
1.9 Scope of Work................. 16
2. Materials and Methods.................................................................................................................................. 18
2.1 Materials ............................................................................................................................................ 18
2.1.1 Instruments and Devices....... 18
2.1.1.1 Centrifuges ............................................................................................................................... 18
2.1.1.2 High Pressure Liquid Chromatography (HPLC) System.......................................................... 18
2.1.1.3 ressure Liquid ChromaHPLC)/Mass Spectrometry (MS) System ................ 18
2.1.1.4 High Pressure Liquid Chromatography (HPLC)/Fluorescence Detection System ................... 19
2.1.1.5 Absorption Spectrometer.......................................................................................................... 19
2.1.1.6 Fluorescence Spectrometer.......................................................................................................19
2.1.1.7 Spectro-electrochemical Cell....................................................................................................19
2.1.1.8 Devices for X-ray Data Collection ........................................................................................... 19
2.1.1.9 Additionally used Instruments and Devices ............................................................................. 20
2.1.2 Chemicals...................................................................................................................................... 20
2.1.3 Computional Support (Software) ..................................................................................................21
2.1.4 Media, Buffers and Stock-Solutions..............................................................................................22
2.1.4.1 Growth Media........................................................................................................................... 22
2.1.4.2 Buffers...................................................................................................................................... 23
2.1.4.3 Stock-Solutions ........................................................................................................................ 23
2.1.5 Strains, Vectors and Oligonucleotides .......................................................................................... 24
2.1.5.1 Strains............... 24
2.1.5.2 Vectors............. 25
2.1.5.3 Oligonucleotides....................................................................................................................... 25
2.2 Methods..................... 26
2.2.1 Microbiological Methods .............................................................................................................. 26
2.2.1.1 Storage and Cultivation of E. coli ............................................................................................ 26
2.2.1.2 Storage and Cultivation of H. salinarum.................................................................................. 26
2.2.1.3 Recording Growth Curves by Optical Density......................................................................... 27
2.2.1.4 dowth Curves by Viable Cell Count ..................................................................... 27
2.2.1.5 Correlation of Optical Density and Internal Cell Volume ........................................................ 27
2.2.2 Molecularbiological Methods........................................................................................................ 28
2.2.2.1 Preparation and Transformation of Electro-Competent E. coli Cells ....................................... 28
2.2.2.2 Preparation and Transformation of Competent H. salinarum Cells ......................................... 28
2.2.2.3 Preparation of Genomic DNA from H. salinarum ................................................................... 29
2.2.2.4 Isolation of Vector-DNA from E. coli...................................................................................... 29
2.2.2.5 Isolation of DNA form Preparative Agarose Gels.................................................................... 29
2.2.2.6 Polymerase Chain Reaction (PCR)........................................................................................... 29
2.2.2.7 Digest of DNA by Restriction Endonulceases.......................................................................... 30
2.2.2.8 Ligation of DNA Fragments..................................................................................................... 30
2.2.2.9 Gel-Electrophoresis of DNA 31
2.2.2.10 Determination of DNA-Concentration ..................................................................................... 31
2.2.2.11 Cloning of Dodecin from H. salinarum.................................................................................... 31
2.2.2.12 Site Directed Mutagenesis of Dodecin from H. salinarum....................................................... 32
2.2.2.13 Cloning of Dodecin from H. halophila 33
2.2.2.14 DNA-Sequencing ..................................................................................................................... 33
2.2.2.15 Isolation of Total RNA from H. salinarum .............................................................................. 34
2.2.2.16 DNase I-Digestion of RNA-samples ........................................................................................ 34
2.2.2.17 Reverse Transcription (RT) and SybrGreen-Based RT-PCR ................................................... 34
2.2.3 Proteinchemical Methods.............................................................................................................. 36
2.2.3.1 SDS-Polyacrylamide Gel-Electrophoresis (SDS-PAGE) of Proteins....................................... 36
2.2.3.2 Electro-Blotting ........................................................................................................................ 37
1 Table of Contents
2.2.3.3 Western Blot Analysis.............................................................................................................. 37
2.2.3.4 Determination of Protein Concentration................................................................................... 38
2.2.3.5 Concentrating Proteins ............................................................................................................. 38
2.2.3.6 Expression and Purification of Dodecin from H. salinarum .................................................... 39
2.2.3.7 Exon and Purification of Dodecin from H. halophila ..................................................... 39
2.2.3.8 Refolding and