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Structure Analysis of DNA Relaxases, the Key Enzymes Of Structure analysis of DNA relaxases, the key enzymes of bacterial conjugation: TraA and its N-terminal relaxase domain of the Gram-positive plasmid pIP501 show specific oriT binding and behave as dimers in solution Vorgelegt von Mgr inz. Jolanta Kopec von der Fakultät für Prozesswissenschaften Der Technischen Universität Berlin Zur Erlangung des akademischen Grades Doktor Ingenieur - Dr. ing. - Promotionsausschuss: Vorsitzender: Prof. Dr. Helmut Görisch 1. Gutachter: Prof. Dr. Ulrich Szewzyk 2. Gutachter: PD Dr. Elisabeth Grohmann 3. Gutachter: Prof. Dr. Walter Keller (Karl-Franzens-Universität, Graz) Tag der wissenschaftlichen Aussprache: 13. Juli 2006 Berlin 2006 D 83 Nothing in the world can take the place of persistence. Talent will not; nothing is more common than unsuccessful people with talent. Genius will not; unrewarded genius is almost a proverb. Education will not; the world is full of educated derelicts. Persistence and determination alone are omnipotent, John Calvin Coolidge, Jr. 2 Parts of the thesis were published in: 1. Jolanta Kopec, Alexander Bergmann, Gerhard Fritz, Elisabeth Grohmann and Walter Keller. TraA and its N-terminal relaxase domain of the Gram-positive plasmid pIP501 show specific oriT binding and behave as dimers in solution. Biochem. J. (2005) 387, 401-409 2. Brigitta Kurenbach, Jolanta Kopec, Marion Mägdefrau, Kristin Andreas, Christine Bohn, Mohammad Yaser Abajy, Walter Keller and Elisabeth Grohmann. The TraA relaxase autoregulates the first putative type IV secretion system from Gram-positive pathogens. Microbiol. (2006) 152, 637-645 3 Acknowledgements I would like to thank all members of Environmental Ecology (Institute of Environmental Technology, Technical University Berlin) and Structural Biology Group (Institute of Chemistry, Karl-Franzens-Universität Graz), especially the group leaders – Prof. Dr. Ulrich Szewzyk and Prof. Dr. Christoph Kratky. I owe particular gratitude to the supervisors of my doctoral thesis, PD Dr. Elisabeth Grohmann and Prof. Dr. Walter Keller for the interesting research subject, their support and for giving me the possibility to work independently. My grateful thanks are due to my colleagues for all the fruitful discussions about science and life: Dr. Monika Oberer, Dr. Brigitta Kurenbach, mgr inż. Andrzej Łyskowski, Mag. Tea Pavkov, Dr. Maria Alexandrino. I also want to thank my family for the support and stamina, they have given me. I want to thank Dipl. Ing. Christine Bohn for letting me be a part of her family during my stay in Berlin. This work would not be possible without the financial support from NaFöG and Berliner Programm zur Förderung der Chancengleichheit für Frauen in Forschung und Lehre. And last, but not least, I express my gratitude to Dipl. Ing. Yvonne Werzer for letting me be a part of her life. 4 Table of contents 1 Introduction ......................................................................................................................10 1.1 Plasmids....................................................................................................................11 1.1.1 pQE expression system.....................................................................................11 1.2 Bacterial cell wall .....................................................................................................13 1.3 Bacterial conjugation................................................................................................14 1.3.1 Conjugation in Gram-negative bacteria............................................................15 1.3.2 Conjugation in Gram-positive bacteria ............................................................17 1.4 Type IV secretion system .........................................................................................17 1.5 Plasmid pIP501.........................................................................................................21 1.6 Origins of transfer of the RSF1010 family...............................................................22 1.7 Relaxases ..................................................................................................................23 2 Aims of the work ..............................................................................................................26 3 Materials ...........................................................................................................................27 3.1 Chemicals .................................................................................................................27 3.2 Kits ...........................................................................................................................28 3.3 Enzymes ...................................................................................................................28 3.4 Protein standards......................................................................................................29 3.5 DNA standards.........................................................................................................30 3.6 Chromatographic resins............................................................................................30 3.7 Instruments ...............................................................................................................31 3.8 Oligonucleotides.......................................................................................................32 3.9 Plasmids and vectors ................................................................................................33 3.10 Microorganisms........................................................................................................33 3.11 PCR programs ..........................................................................................................34 3.12 Buffers ......................................................................................................................34 3.13 Media........................................................................................................................34 4 Methods ............................................................................................................................35 4.1 DNA methods...........................................................................................................35 4.1.1 Preparative plasmid isolation...........................................................................35 4.1.2 Cloning .............................................................................................................35 4.1.2.1 Cloning of wild type traA.............................................................................35 4.1.2.2 Cloning of traAN268 ......................................................................................36 4.1.2.3 Cloning of traAN246 ......................................................................................36 5 4.1.3 Plasmid DNA mini-prep...................................................................................37 4.1.4 Isolation of plasmid RSF 1010 .........................................................................37 4.1.5 Preparation of hairpin_nic oligonucleotide for crystallization trials................38 4.2 Microbiological methods..........................................................................................38 4.2.1 Preparation of E. faecalis JH2-2 (pIP501) cell lysate ......................................38 4.2.2 Preparation and transformation of E. coli BL21(DE3) competent cells...........38 4.2.3 Preparation of E. coli XL1Blue competent cells ..............................................39 4.2.4 Transformation of E. coli XL1Blue competent cells........................................39 4.3 Screening of transformants.......................................................................................39 4.3.1 PCR test............................................................................................................39 4.3.2 Restriction analysis...........................................................................................40 4.3.3 DNA labeling....................................................................................................40 4.4 Biochemical methods ...............................................................................................40 4.4.1 TraA, TraAN268 and TraAN246 expression .......................................................40 4.4.2 TraA, TraAN268 and TraAN246 purification......................................................41 4.4.3 Orf7 constructs expression and purification.....................................................42 4.4.3.1 GST-Orf7∆TM .............................................................................................42 4.4.3.2 7His-Orf7......................................................................................................42 4.4.3.3 MBP-Orf7∆TM ............................................................................................42 4.4.4 Zymogram analysis..........................................................................................43 4.4.5 Crystallization trials..........................................................................................43 4.4.5.1 Vapor diffusion.............................................................................................43 4.4.5.2 Microbatch....................................................................................................43 4.5 Biophysical methods ................................................................................................44
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