Integration of redox and light signals by the regulator protein AppA in Rhodobacter sphaeroides [Elektronische Ressource] / vorgelegt von Yuchen Han

Sujets

• Biologie

Informations

• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2006
• Nombre de lectures: 22
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

Integration of redox and light signals by
the regulator protein AppA in
Rhodobacter sphaeroides




Inaugural-Dissertation
zur Erlangung
des
Doktorgrades der Naturwissenschaften
(Dr. rer. nat.)



vorgelegt von


M.Sc.- Biol. Yuchen Han


aus
Jiangsu, P.R. China




angefertigt am Institut für Mikrobiologie und Molekularbiologie
Fachbereich Biologie und Chemie
Justus-Liebig-Universität Giessen




Giessen, Oktober 2006






Die vorliegende Arbeit wurde angefertigt am Institut für Mikrobiologie und
Molekularbiologie des Fachbereiches 08 der Justus-Liebig-Universität Giessen in der Zeit
von September 2002 bis Oktober 2006 unter der Leitung von Prof. Dr. Gabriele Klug.





























1. Gutachterin: Prof. Dr. Gabriele Klug

Institut für Mikrobiologie und Molekularbiologie
Justus-Liebig-Universität Giessen

2. Gutachter: Prof. Dr. Rainer Renkawitz
Institut für Genetik iebig-Universität Giessen


Contents


Abbreviations ................................................................................................ v
Publications ................................................................................................. vii

1 Introduction.............................................................................................. 1

1.1 Blue light photoreceptors.................1
1.1.1 LOV domain proteins..................................................................................................................1
1.1.2 The photolyase/cryptochrome family..........................................................................................2
1.1.3 Photoactive yellow protein (PYP)...............................................................................................3
1.1.4 BLUF domain proteins................................................................................................................4
1.2 Phylogenetics and physiology of Rhodobacter sphaeroides............................................................5
1.3 Blue light photoreceptors in Rhodobacter sphaeroides...................................................................6
1.4 Regulation of photosynthesis genes by light and oxygen in Rhodobacter sphaeroides ................7
1.4.1 The photosynthetic apparatus in R. sphaeroides.........................................................................7
1.4.2 Control of photosynthesis genes expression in R. sphaeroides...................................................9
1.4.2.1 The PrrB/PrrA two-component system .............................................................................11
1.4.2.2 The AppA/PpsR antirepressor/repressor system ...............................................................12
1.4.2.3 Anaerobic regulator FnrL..................................................................................................14
1.4.2.4 The puf-binding protein Spb..............................................................................................15
1.4.2.5 Thioredoxin (Trx)..............................................................................................................15
1.4.2.6 Other factors in photosynthesis genes expression .............................................................16
1.5 Objectives of this work...................................................................................................................16

2 Materials ................................................................................................. 18

2.1 Chemicals and reagents..................................................................................................................18
2.2 Enzymes...........................................................................................................................................19
2.3 Commercial reaction buffers .........................................................................................................20
2.4 Antibiotics........................................................................................................................................20
2.5 Molecular biological kits ................................................................................................................20
2.6 Antibodies........................................................................................................................................20
2.7 Strains............................21
2.8 Plasmids...........................................................................................................................................22
2.9 Oligonucleotides..............................................................................................................................23
2.10 Other materials and equipments ...................................................................................................27

3 Methods................................................................................................... 29

3.1 Microbiological methods ................................................................................................................29
3.1.1 Cultivation of E. coli29
3.1.1.1 E. coli plating culture ........................................................................................................29
3.1.1.2 E. coli liquid culture..........................................................................................................29
3.1.2 Cultivation of R. sphaeroides....................................................................................................29
3.1.2.1 R. sphaeroides plating culture...........................................................................................30
- i -
3.1.2.2 R. sphaeroides liquid culture.............................................................................................30
3.1.2.3 Blue light-shift experiments under semi-aerobic conditions .............................................30
3.1.2.4 Oxygen-shift experiment...................................................................................................31
3.1.3 Preparation of glycerol stocks for the -80°C strain collection ..................................................31
3.2 DNA preparation ............................................................................................................................31
3.2.1 Plasmid minipreparation by alkaline lysis ................................................................................31
3.2.2 Plasmid midipreparation ...........................................................................................................32
3.2.3 Chromosomal DNA isolation......32
3.2.4 Gel extraction............................................................................................................................33
3.2.5 Gel electrophoresis of DNA......................................................................................................33
3.3 Molecular cloning ...........................................................................................................................34
3.3.1 Polymerase chain reaction (PCR) .............................................................................................34
3.3.1.1 Standard PCR...........34
3.3.1.2 Site-specific mutagenesis by overlap extension ................................................................34
3.3.1.3 PCR-based random mutagenesis .......................................................................................34
3.3.2 Restriction.................................................................................................................................35
3.3.3 Ligation.....................................................................................................................................35
3.3.3.1 Standard ligation ...............................................................................................................35
3.3.3.2 Ligation using the pGEX®-T vector.................................................................................35
3.3.3.3 Ligation using the pDrive vector.......................................................................................36
3.3.4 Preparation of E. coli competent cells for electroporation........................................................36
3.3.5 Transformation by electroporation............................................................................................36
3.4 Extraction, purification and analysis of mRNA from R. sphaeroides.........................................37
3.4.1 RNA isolation ...........................................................................................................................37
3.4.1.1 Hot-phenol extraction........................................................................................................37