The transmembrane signaling in chimeras of the E.coli chemotaxis receptors and bacterial class III adenylyl cyclases [Elektronische Ressource] / vorgelegt von Kajal Kanchan

Walter Wheeler Cook

Sujets

• Naturwissenschaften

Informations

• Publié par: eberhard_karls_universitat_tubingen
• Publié le: 01 janvier 2011
• Nombre de lectures: 19
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Transmembrane Signaling in Chimeras of the E. coli
Chemotaxis Receptors and Bacterial Class III
Adenylyl Cyclases









Dissertation
der Mathematisch-Naturwissenschaftlichen Fakultät
der Eberhard Karls Universität Tübingen
zur Erlangung des Grades eines
Doktors der Naturwissenschaften
(Dr. rer. nat.)






vorgelegt von
Kajal Kanchan
Jamshedpur, India



Tübingen
2011




























Tag der mündlichen Qualifikation: 04.02.2011
Dekan: Prof. Dr. Wolfgang Rosenstiel
1. Berichterstatter: Prof. Dr. Joachim E. Schultz
2. Berichterstatter: Prof. Dr. Klaus Hantke Acknowledgement


The experimental part of this thesis work was performed within the period of October 2006 to
January 2011 under the supervision of Prof. Dr. J.E. Schultz.

I would like to thank my supervisor, Prof. Dr. J. E. Schultz, whose encouragement, guidance and
support from the initial to the final level enabled me to develop an understanding of the project
as well as finish my PhD work successfully. This thesis work would not have been possible
without his guidance.

I would like to thank Prof. Dr. Klaus Hantke for taking part in the evaluation of my thesis as well
as his help in my project by performing several in vivo experiments. You never hesitated to help
me whenever I approached you.
It is an honour to have Prof. Peter Ruth, Priv. Doz. Dr. Martina Düfer, Prof. Dr. Klaus Hantke
and Prof Dr. J. E. Schultz for my thesis examination.
I am grateful to my supervisor in India, Prof. Dr. Sandhya Visweswariah for recommending me
to Prof. Dr. J.E. Schultz.
I owe my deepest gratitude to my friend Manoj Kumar and Janani Natarajan for reading my
thesis and their suggestions.
I would like to thank my colleagues, Karin, Laura, Ana, Janani, Yinglan, Franziska Berndt,
Jürgen Linder and Iman Mansi for their encouragements and constant support in terms of lab
work as well as productive discussions. Karin I am grateful to you for writing the German
version of my thesis abstract as well as I am thankful to Laura for helping me filling all the forms
during my thesis submission. I would also like to thank Ms. Ursula Kurz for her technical
support which kept the lab running smoothly. I would like to thank Mrs. Anita Schultz for
helping me in cloning most of my difficult constructs. At last but not the least I would like to thank my parents, my brother and sister, all my friends in
Germany as well as back in India. Without their encouragement and best wishes I would never
have been able to finish my work successfully.


TABLE OF CONTENT

TABLE OF CONTENT........................................................................................................... I
ABBREVIATIONS............................................................................................................... VI
1. INTRODUCTION1
1.1 SIGNAL TRANSDUCTION ....................................................................................................1
1.2 ADENYLYL CYCLASES ......................................................................................................1
1.2.1 Mammalian Adenylyl Cyclases ................................................................................2
1.2.2 Bacterial Adenylyl Cyclases.....................................................................................3
1.2.2.1 Mycobacterium tuberculosis cyclases................................................................4
1.2.2.2 Cyanobacterial Adenylyl cyclases .....................................................................6
1.3 BACTERIAL CHEMOTAXIS RECEPTORS ..............................................................................7
1.4 AIM...................................................................................................................................9
2. MATERIALS AND METHODS ......................................................................................11
2.1 CHEMICALS.....................................................................................................................11
2.2 EQUIPMENTS...................................................................................................................12
2.3 BUFFERS AND SOLUTIONS:..............................................................................................12
2.3.1 Molecular Biology buffers and solutions................................................................12
2.3.2 Protein chemistry buffers and solutions..................................................................13
2.4 BACTERIAL STRAINS .......................................................................................................15
2.5 PLASMIDS .......................................................................................................................16
2.6 OLIGONUCLEOTIDES16
2.6.1 Cloning Primers ......................................................................................................17
2.7 MOLECULAR BIOLOGY METHODS....................................................................................24
2.7.1 Polymerase chain reaction24
2.7.2 Agarose electrophoresis........................................................................................25
2.7.3 Photometric determination of DNA concentration...............................................25
2.7.4 Enzymatic digestion..............................................................................................25
2.7.5 Generation of blunt ends.......................................................................................26
2.7.6 5’-phosphorylation of PCR products ....................................................................26
2.7.7 Dephosphorylation of plasmid vectors .................................................................26
I

2.7.8 Ligation of the DNA fragment.............................................................................26
2.7.9 Transformation of recombinant DNA..................................................................26
2.7.10 Isolation and Purification of DNA.......................................................................27
2.7.11 DNA sequencing..................................................................................................27
2.7.12 Permanent cultures...............................................................................................28
2.7.13 Cloning.................................................................................................................28
2.7.13.1 Cloning of CyaG and CyaG chimeras ...........................................................28
2.7.13.2 Periplasmic Binding proteins (MBP, GBP, DBP) .........................................32
2.7.13.3 Cloning of Rv3645 chimeras .........................................................................32
2.7.13.4 Cloning of Rv1264 chimeras33
2.7.13.5 Cloning of CyaB1 chimeras...........................................................................33
2.7.13.6 Cloning of Rv1625c chimeras .......................................................................34
2.7.13.7 Chimeras with swapped transmembranes and ligand binding domains ........37
2.7.13.8 Transmembrane deletion chimeras ................................................................39
2.7.13.9 Tsr and Tar receptor mutant chimeras ...........................................................39
2.8 PROTEIN CHEMISTRY METHODS......................................................................................41
2.8.1 Preculture .............................................................................................................41
2.8.2 Expression41
2.8.3 Purification of soluble proteins from E. coli........................................................41
2.8.4 Preparation of membrane fractions from E. coli..................................................41
2.8.5 Solubilization of the membrane protein...............................................................42
2.8.6 Purification of periplasmic protein by osmotic shock .........................................42
2.8.7 Bio-Rad Protein determination ............................................................................42
2.8.8 Densitometry for protein determination...............................................................43
2.8.9 Bioinformatics......................................................................................................43
2.8.10 SDS-PAGE ..........................................................................................................43
2.8.11 Western Blot ........................................................................................................44
2.8.12 Adenylyl cyclase assay ........................................................................................44
3. RESULTS ...........................................................................................................................46
3.1 HAMP MEDIATED SIGNAL TRANSDUCTION BY A CLASS IIIA AC ..........