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Development of Bacillus Subtilis Spores and Cells for Surface Display of Proteins

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Development of Bacillus Subtilis Spores and Cells for Surface Display of Proteins Development of Bacillus subtilis spores and cells for surface display of proteins Dissertation zur Erlangung des Grades eines -Doktors der Naturwissenschaften- der Fakultät für Biologie, Chemie und Geowissenschaften der Universität Bayreuth vorgelegt von Nguy ễn Qu ỳnh Anh Bayreuth 2010 Die vorliegende Arbeit wurde in der Zeit von August 2007 bis Oktober 2010 an der Universität Bayreuth am Lehrstuhl für Genetik unter der Betreuung von Prof. Dr. Wolfgang Schumann angefertigt. Vollständiger Abdruck der von der Fakultät für Biologie, Chemie und Geowissenschaften der Universität Bayreuth genehmigten Dissertation zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften (Dr. rer. nat.) Promotionsgesuch eingereicht am: 15.12.2010 Tag des wissenschaftlichen Kolloquiums: 11.03.2011 Erstgutachter: Prof Dr. Wolfgang Schumann Zweitgutachter: PD. Dr. Steffen Kolb Vorsitzender: Prof. Dr. Konrad Dettner Prof. Dr. Franz G. Meussdoerffer Prof. Dr. Birgitta Wöhrl To my parents Acknowledgements First of all, I would like to express my sincere gratitude to my supervisor Prof. Dr. Wolfgang Schumann for his patience, enthusiasm, continuous guidance, and encouragement throughout my research. I also heartily appreciate his substantial support during my years in Bayreuth. I am thankful for Prof. Dr. Thomas Wiegert, whose valuable advices and discussions helped me a lot throughout my working process. Many thanks are due to Prof. Dr. Olaf Stemmann and PD. Dr. Stefan Heidmann for providing facilities for my study. My special thanks also go to Markus Hermann for his help in working with microscope and FACS. My sincere thanks go to Prof. Dr. Junehyung Kim in Dong-A University, Busan, South Korea for his valuable advices and friendly help. I would like to thank Karin Angermann and Petra Helies for their valuable assistance and making a warm atmosphere in the Lab. I would like to thank all the lab members of Prof. Stemmann and Dr. Heidmann for their help in experiments and discussions. Special thanks are due to Derrick and Thierry (Ù) for the critical reading of my thesis manuscript. My deepest appreciations are due to all my colleagues: Hu ệ, Kelly and Kati for their numerous advices, the friendly cooperation and for making my stay in Bayreuth unforgettable. Special thanks go to Dr. Markus Helfrich for his considerable suggestions and discussions. I would like to thank my dear friends ch ị Trinh, ch ị H ường, Minh and my other country-mates for their support and inestimable friendship. My deepest thanks and gratitude go to Seungchul, Sonal, Johannes and Kristin. I thank all my friends in Bayreuth for the support and friendship that made my stay here in Germany so fantastic. I thank the Bayerische Forschungsstiftung and the Frauenbeauftragten in University of Bayreuth for the financial support. Finally, I wish to express my love and gratitude to my family in Viet Nam. Many thanks are due to my parents for giving birth to me, raising me up, unconditionally loving and supporting me throughout my life. To them I dedicate this thesis. I would like to thank my brother and sister- in-law for their love, support and untiring encouragement. They are my best pals. Contents Contents Zusammenfassung................................................................................................................ 1 Summary ............................................................................................................................... 3 1 Introduction ................................................................................................................... 5 1.1 Microbial surface display ...................................................................................................... 5 1.1.1 Phage display ......................................................................................................................... 5 1.1.2 Bacterial surface display........................................................................................................ 6 1.1.2.1 Cell surface display in Gram-negative bacteria ................................................................ 7 1.1.2.2 Cell surface display in Gram-positive bacteria ................................................................. 9 1.1.2.3 Bacterial spore surface display ....................................................................................... 11 1.1.3 Surface display in yeast ....................................................................................................... 12 1.1.4 Applications of microbial surface display ........................................................................... 13 1.2 Bacillus subtilis spore ........................................................................................................... 17 1.2.1 Sporulation in B. subtilis ..................................................................................................... 17 1.2.2 Spore morphology ............................................................................................................... 20 1.2.3 The spore coat compartment ............................................................................................... 22 1.2.4 Regulation of spore coat protein genes ............................................................................... 27 1.2.5 Model for coat assembly ..................................................................................................... 29 1.3 Intein and protein splicing .................................................................................................. 33 1.3.1 Configuration of the intein .................................................................................................. 33 1.3.2 Mechanism of protein splicing ............................................................................................ 35 1.3.3 Engineered inteins and mini-inteins used in protein purification ........................................ 38 1.4 Cellulases and their applications ........................................................................................ 39 i Contents 1.4.1 Cellulose and cellulose degradation enzymes .................................................................... 39 1.4.2 Applications of cellulases .................................................................................................... 42 1.5 Aims of the thesis ................................................................................................................. 44 2 Materials and methods ............................................................................................... 45 2.1 Materials ............................................................................................................................... 45 2.1.1 Bacterial strains ................................................................................................................... 45 2.1.2 Plasmids ............................................................................................................................... 46 2.1.3 Oligonucleotides .................................................................................................................. 49 2.1.4 Antibiotics ........................................................................................................................... 51 2.1.5 Enzymes .............................................................................................................................. 51 2.1.6 Antibodies............................................................................................................................ 52 2.1.7 Media ................................................................................................................................... 52 2.1.8 Chemicals and biochemicals ............................................................................................... 52 2.1.9 Kits ...................................................................................................................................... 53 2.2 Methods ................................................................................................................................. 53 2.2.1 General methods .................................................................................................................. 53 2.2.1.1 PCR ................................................................................................................................. 53 2.2.1.2 Cloning ........................................................................................................................... 53 2.2.1.3 Growth and collection of samples .................................................................................. 54 2.2.1.4 Spore purification method .............................................................................................. 54 2.2.2 Protein methods ................................................................................................................... 55 2.2.2.1 Extraction of denatured total cellular lysate from B. subtilis ......................................... 55 2.2.2.2 Preparation of proteins from the B. subtilis cell wall fraction ........................................ 55 2.2.2.3 Extraction of B. subtilis spore-coat proteins ................................................................... 55 ii Contents 2.2.2.4 Measurement of protein concentrations .......................................................................... 56 2.2.2.5 Protein electrophoresis
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