İSTANBUL TECHNICAL UNIVERSITY INSTITUTE OF SCIENCE AND TECHNOLOGY IN VIVO EVOLUTIONARY ENGINEERING OF A BORON-RESISTANT BACTERIUM: Bacillus boroniphilus M.Sc. Thesis by Mustafa ŞEN Department : Advanced Technologies Programme : Molec. Biol. – Gen. & Biotechno. Thesis Supervisor: Assoc. Prof. Dr. Zeynep Petek ÇAKAR JANUARY 2010 ISTANBUL TECHNICAL UNIVERSITY INSTITUTE OF SCIENCE AND TECHNOLOGY IN VIVO EVOLUTIONARY ENGINEERING OF A BORON-RESISTANT BACTERIUM: Bacillus boroniphilus M.Sc. Thesis by Mustafa ŞEN (521071044) Date of submission : 25 December 2009 Date of defence examination: 29 January 2010 Supervisor (Chairman) : Assoc. Prof. Dr. Z. Petek ÇAKAR (ITU) Members of the Examining Committee : Prof. Dr. Tülay TULUN (ITU) Assis. Prof. Dr. Fatma Neşe KÖK (ITU) JANUARY 2010 İSTANBUL TEKNİK ÜNİVERSİTESİ FEN BİLİMLERİ ENSTİTÜSÜ IN VIVO EVRİMSEL MÜHENDİSLİK YÖNTEMİYLE BORA DİRENÇLİ Bacillus boroniphilus BAKTERİSİNİN GELİŞTİRİLMESİ YÜKSEK LİSANS TEZİ Mustafa ŞEN (521071044) Tezin Enstitüye Verildiği Tarih : 25 Aralık 2009 Tezin Savunulduğu Tarih : 29 Ocak 2010 Tez Danışmanı : Doç. Dr. Z. Petek ÇAKAR (İTÜ) Diğer Jüri Üyeleri : Prof. Dr. Tülay TULUN (İTÜ) Yrd. Doç. Dr. Fatma Neşe KÖK (İTÜ) OCAK 2010 FOREWORD I want to thank my supervisor, Assoc. Prof. Zeynep Petek Çakar, for her great help, invaluable guidance and contribution during the development of this thesis. I specially thank to Ülkü Yılmaz for her sincerity, collaboration and endless help during the experiments. It was a pleasure for me to work with her. I have learnt so much from her. I would like to thank to Prof. Dr. Süleyman Akman and Res. Assist. Aslı Ege from ITU Chemistry Department for providing the FAAS analysis and their technical support. I would also like to thank Ali Dinler of being so supportive with the necessary hardware and software. I am so thankful to Burcu Turanlı and Tugba Aloglu for their guidance and patience. I would like to thank my mother Emriye Şen and my father Selahattin Şen, my brothers Bayram Ali Şen, Uğur Şen, Abdurrahim Şen and my sister Nurgül Şen for their endless patience, support and endless reliance. Lastly, I am grateful to National Boron Research Institute for the financial support. January 2010 Mustafa ŞEN Molecular Biologist v vi TABLE OF CONTENTS Page ABBREVIATIONS ................................................................................................... ix LIST OF TABLES .................................................................................................... xi LIST OF FIGURES ................................................................................................ xiii SUMMARY .............................................................................................................. xv ÖZET ....................................................................................................................... xvii 1. INTRODUCTION .................................................................................................. 1 1.1 Bacillus Species .................................................................................................. 1 1.1.1 Brief information. ……………………………………….........…….........1 1.1.2 Scientific & industrial importance of Bacillus spp. ...................................3 1.2 Why Boron? ....................................................................................................... 5 1.3 Boron Resistance and Metabolism of Plants and Microorganism …………….8 1.3.1 Plant studies ……………………………………………………………..8 1.3.2 Cyanobacteria, azotobacteria and diatom studies ……………….……..10 1.3.3 Bacillus boroniphilus …………………………………………………..12 1.4 How to Obtain Boron Resistant Bacillus boroniphilus .……………………...14 1.4.1 Metabolic engineering ....……………………………………………….14 1.4.2 An inverse metabolic engineering strategy: evolutionary engineering ...16 1.5 The Aim of the Present Study .......................................................................... 17 2. MATERIAL AND METHODS ........................................................................... 19 2.1 Materials, Equipment and Organisms .............................................................. 19 2.1.1 Software, programs and websites ..........................................................19 2.1.2 Bacillus boroniphilus strain ....................................................................19 2.1.3 Bacteria culture media.............................................................................19 2.1.3.1 Composition of medium 220 ......................................................19 2.1.4 Chemicals, buffers and solutions ............................................................19 2.1.5 Laboratory equipment .............................................................................20 2.2 Methods ............................................................................................................ 21 2.2.1 Growth curve ……………………………………………….................21 2.2.2 Ethyl methane sulphonate (EMS) mutation ............................................21 2.2.3 General boron stress screening and determination of initial boron concentration for generation obtaining ...................................................21 2.2.4 Obtaining increasing stress generations and stock culture preparation .......................................................................22 2.2.5 Selection of individual mutants ………………………..……………....23 2.2.6 Characterization of individual mutants .……….……………………….24 2.2.6.1 Determination the most resistant individuals by using general screening strategy..........................................24 2.2.6.2 Determination of cross-resistance to other stress conditions ….24 2.2.6.3 Principle component analysis ……………………………….…26 2.2.6.4 Determination of boron content of individuals by AAS ............27 2.2.7 Transcriptomic analysis ............................................................................ 28 vii 2.2.7.1 Primer design ............................................................................28 2.2.7.2 RNA isolation ...........................................................................28 2.2.7.3 cDNA synthesis ........................................................................29 2.2.7.4 PCR and optimization steps ......................................................29 3. RESULTS .............................................................................................................. 33 3.1 Growth Curve ................................................................................................... 33 3.2 EMS Mutated Populations ................................................................................ 34 3.3 Screening of the Wild-type for Determination of Initial Boron Sterss Level ........................................................ ………………35 3.4 Obtaining Generations Through Increasing Stress Strategy and Determination of Boron Resistance ........................................................... 36 3.5 Selection of Individual Mutants and Determination of the Most Resistance Individuals ................................................................... 38 3.6 Characterization of Selected Individuals .......................................................... 40 3.6.1 Cross resistance tests of individual mutants and wild-type ....................40 3.6.1.1 Chromium stress ........................................................................40 3.6.1.2 Zinc stress ..................................................................................40 3.6.1.3 Cobalt stress ...............................................................................41 3.6.1.4 Copper stress ..............................................................................41 3.6.1.5 Iron stress ...................................................................................43 3.6.1.6 Osmotic stress ............................................................................44 3.6.1.7 Ethanol stress .............................................................................45 3.6.1.8 Oxidative stress ..........................................................................46 3.6.1.9 Sorbitol stress .............................................................................47 3.6.1.10 Heat stress ................................................................................47 3.6.1.11 Freeze-thaw stress.....................................................................48 3.6.2 Analysis of cross-resistance test results .................................................49 3.6.1.1 Analysis via Excel charts ...........................................................49 3.6.1.2 Analysis via principle component analysis (PCA) program …..50 3.6.3 Boron contents of individuals measured by AAS ……………………..52 3.7 Transcriptomic Analysis …...............................................................................53 4. DISCUSSION & CONCLUSION ....................................................................... 57 REFERENCES ......................................................................................................... 63 APPENDICES .......................................................................................................... 67 CURRICULUM VITAE .......................................................................................... 73 viii ABBREVIATIONS PCA : Principle Component Analysis AAS : Atomic Absorption Spectrometer EMS : Ethyl Methane Sulphonate H3BO3 : Boric Acid DAP : Meso-Diaminopimelic Acid RG-II : Rhamnogalacturonan-II OD : Optical