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Concentrating Mechanisms and Development of Molecular Tools for Chlamydomonas Reinhardtii University of Nebraska - Lincoln DigitalCommons@University of Nebraska - Lincoln Theses and Dissertations in Biochemistry Biochemistry, Department of 12-2013 Transcriptomic Analyses of the CO2-Concentrating Mechanisms and Development of Molecular Tools for Chlamydomonas reinhardtii Andrew J. Brueggeman University of Nebraska-Lincoln, [email protected] Follow this and additional works at: https://digitalcommons.unl.edu/biochemdiss Part of the Biochemistry Commons, and the Molecular Biology Commons Brueggeman, Andrew J., "Transcriptomic Analyses of the CO2-Concentrating Mechanisms and Development of Molecular Tools for Chlamydomonas reinhardtii" (2013). Theses and Dissertations in Biochemistry. 13. https://digitalcommons.unl.edu/biochemdiss/13 This Article is brought to you for free and open access by the Biochemistry, Department of at DigitalCommons@University of Nebraska - Lincoln. It has been accepted for inclusion in Theses and Dissertations in Biochemistry by an authorized administrator of DigitalCommons@University of Nebraska - Lincoln. TRANSCRIPTOMIC ANALYSES OF THE CO2-CONCENTRATING MECHANISM AND DEVELOPMENT OF MOLECULAR TOOLS FOR CHLAMYDOMONAS REINHARDTII by Andrew J Brueggeman A Dissertation Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Doctor of Philosophy Major: Biochemistry Under the Supervision of Professor Donald P. Weeks Lincoln, Nebraska December, 2013 TRANSCRIPTOMIC ANALYSES OF THE CO2-CONCENTRATING MECHANISM AND DEVELOPMENT OF MOLECULAR TOOLS FOR CHLAMYDOMONAS REINHARDTII Andrew J Brueggeman, Ph.D. University of Nebraska, 2013 Advisor: Donald P. Weeks Microalgae, such as Chlamydomonas reinhardtii, account for a large percentage of photosynthesis that occurs on the planet. Many algae possess a Carbon- Concentrating Mechanism, or CCM, that actively transports inorganic carbon (Ci) into the cell to create artificially high internal levels of CO2, enhancing their rate of carbon fixation. The production of biofuels from algal sources can serve as both a renewable and carbon-neutral energy source. This thesis details research in Chlamydomonas, in the effort to both better understand the CCM in algae and improve laboratory and industrial manipulations with algae. In the first chapter of this thesis, changes in the Chlamydomonas transcriptome in response to changes in environmental CO2 levels are investigated. In addition to witnessing changes in the transcription of 38% of Chlamydomonas genes, numerous patterns of genetic response are detailed, including a transient reduction in overall transcription that is later alleviated as cells became acclimated to the new environment. Our data also reveal a vast bidirectional promoter system in Chlamydomonas, detail markedly different responses of gene families, and elucidated several putative binding motifs for transcriptional regulators involved in responses to low CO2. The remaining two chapters detail efforts to develop tools for use in Chlamydomonas. In the second chapter, drawing upon prior knowledge of herbicide resistance in higher plants, we developed several herbicide-resistant genes for optimal function in Chlamydomonas. As a result, we now have resistance markers for three classes of herbicides. These genes can be used either as selectable markers in transgenic experiments or as tools for maintaining axenic cultures in large-scale biofuel production facilities. The final chapter details efforts to develop an in-house method of synthesizing Transcription Activator-Like Effector (TALE) proteins to target specific DNA sequences in Chlamydomonas. Final TALE constructs were fused with FokI nuclease domains to create TAL Effector Nucleases (TALENs) that can create DNA double-strand breaks at specific sites. With an efficient method of synthesizing TALEN genes de novo, efforts can proceed to utilize this technology to create specific modifications within the Chlamydomonas genome. iv Table of Contents TRANSCRIPTOMIC ANALYSES OF THE CO2-CONCENTRATING MECHANISM AND DEVELOPMENT OF MOLECULAR TOOLS FOR CHLAMYDOMONAS REINHARDTII Abstract Table of Contents ........................................................................................................... iv Dedication ..................................................................................................................... vii Acknowledgements ...................................................................................................... viii List of Figures .................................................................................................................. x List of Abbreviations ...................................................................................................... xii CHAPTER 1 - Literature Review ........................................................................................1 CHAPTER 1.1 - Chlamydomonas reinhardtii CCM..................................................2 References ..................................................................................................19 CHAPTER 1.2 - Herbicides ...................................................................................26 References ..................................................................................................38 CHAPTER 1.3 - TAL Effectors ...............................................................................43 References ..................................................................................................62 CHAPTER 2 - Activation of the Carbon Concentrating Mechanism by CO2 Deprivation Coincides with Massive Transcriptional Restructuring in Chlamydomonas reinhardtii....68 v Abstract .............................................................................................................69 Introduction .......................................................................................................70 Results and Discussion .......................................................................................72 Methods and Materials .................................................................................... 110 References ....................................................................................................... 119 CHAPTER 3 - Development of Herbicide-Resistance Technologies for Use in the Green Alga Chlamydomonas reinhardtii ................................................................................. 126 Abstract ........................................................................................................... 126 Introduction ..................................................................................................... 128 Results ............................................................................................................. 132 Discussion ........................................................................................................ 153 Methods and Materials .................................................................................... 154 References ....................................................................................................... 161 CHAPTER 4 - Synthesis of Custom-Designed TAL Effector Nuclease (TALEN) Genes for Targeted Gene Modifications in Chlamydomonas reinhardtii ....................................... 164 Abstract ........................................................................................................... 164 Introduction ..................................................................................................... 166 Results ............................................................................................................. 167 vi Discussion ........................................................................................................ 177 Methods and Materials .................................................................................... 177 References ....................................................................................................... 185 APPENDIX A - Supplemental Material from Chapter Two ............................................. 207 vii Dedication I would like to dedicate this thesis to my grandfather, George H. Johnson, who, just as I do, held a passion for both science and agriculture. It is my hope and prayer that you will fully realize what a wonderful world this is; that you will try to understand, in so far as you can, how things work and why. So many, if not all, things around us are governed by scientific or natural laws and so can be understood. -George Henry Johnson, Personal Memoirs, 1910 - 2004 viii Acknowledgements I would like to acknowledge the support given to me by my advisor, Dr. Donald Weeks, during my time in graduate school. He has been instrumental in helping me develop the skills and knowledge to succeed as a scientist. I also would like to thank members of the Weeks lab, including Kempton Horken, Wen Zhi Jiang, Amy Knobbe, & Thomas Plucinak, for taking the time to answer questions and critique ideas I have had. Much of my computational work has been assisted by Dr. Istvan Ladunga, and I would like to thank him for taking time to teach me the skills needed for much of my work. In addition, I appreciate the guidance provided by all members of my committee, Dr. Weeks, Dr. Ladunga, Dr. Becker, Dr. Fromm, and Dr. Riekhof, for their support and scientific guidance as I proceeded through my degree. The University of Nebraska has been my home for
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