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A Study of the Impact of Mazie SBE I on the [Alpha]-Polyglucan Produced in Synechocystis Sp

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A Study of the Impact of Mazie SBE I on the [Alpha]-Polyglucan Produced in Synechocystis Sp Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 2007 A study of the impact of mazie SBE I on the [alpha]- polyglucan produced in Synechocystis sp. strain PCC 6803 Shayani Deborah Nesaranjani Pieris Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Part of the Plant Biology Commons Recommended Citation Pieris, Shayani Deborah Nesaranjani, "A study of the impact of mazie SBE I on the [alpha]-polyglucan produced in Synechocystis sp. strain PCC 6803" (2007). Retrospective Theses and Dissertations. 15850. https://lib.dr.iastate.edu/rtd/15850 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. A study of the impact of mazie SBE I on the -polyglucan produced in Synechocystis sp. strain PCC 6803 by Shayani Deborah Nesaranjani Pieris A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Plant Physiology Program of Study Committee: Martin H. Spalding, Major Professor Madan K. Bhattacharyya Jay -lin Jane David J. Oliver Paul M. Scott Iowa State University Ames, Iowa 200 7 Copyright © Shayani Deborah Nesara njani Pieris , 200 7. All right s reserved. UMI Number: 3294976 UMI Microform 3294976 Copyright 2008 by ProQuest Information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. ProQuest Information and Learning Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, MI 48106-1346 ii This dissertation is dedicated to my Savior and Lord, Jesus Christ, who is the Creator of the starch granule. To Him be the glory for His awesome works. The order and grandeur of creation testifies to His gre at love and goodness! iii TABLE OF CONTENTS ACKNOWLEDGEMENTS v PREFACE vi ABSTRACT ix CHAPTER 1: INTRODUCTION /L ITERATURE REVIEW 1 Biosynthesis 1 Starch and g lycogen o verview 2 Starch 2 Glycogen 3 Starch structure 4 Amylopectin structure 7 Enzymes involved in g lycogen metabolism in Synechocystis 9 Synthesis/Anabolism 9 Breakdown /Catabolism 10 Enzymes involved in st arch/glycogen biosynthesis 10 1) Branching enzymes 10 2) Starch synthases 18 3) Star ch d ebranching enzymes 19 4) Dis proportionating enzyme 20 Current models explaining granule f ormation and our H ypothesis 21 1) A p hysical process 21 2) Th e glucan trimming model 22 3) Prote in protein interaction 22 4) Water soluble polysaccharide clearing model 22 5) Two -step branching an d improper branch clearing model 23 Our Hypothesis 23 Hypothesis tested in this dissertation 24 Th e cyanobacterial system 24 Advantages and disadvantages in usi ng the cyanobacterial system 25 Photosynthesis in Synechocystis sp. Strain PCC 6803 27 References 27 CHAPTER 2: NEED FOR GLYCOGEN BIOSYNTHESIS IN Synechocystis sp. PCC 6803 AND FURTHER CHARACTERIZATION OF THE M3 MUTANT LACKING GLYCOGEN BRANCHING ENZYME .. 36 Abstract 36 Introduction 37 Materials & Methods 38 Mutant construction 38 PCR analysis 40 Growh condition and growth curve s 41 iv Glucan extraction, amyloglucosidase digestion and HPSEC analysis 42 Soluble protein extraction and enxyme assay 43 Gel permeatio n chromatography (GPC) and high -performance anion exchange chromatography (HPAEC) analysis 44 Results 45 Discussion 59 References 63 CHAPTER 3 : EFFECT OF MAIZE SBE I ON THE NATURE OF THE ALPHA POL YGLUCAN PRODUCED IN Synechocystis sp. PCC 6803 65 Abstract 65 Introduction 66 Materials & Methods 68 Mutant construction 68 PCR analysis 69 Growh condition and growth curve s 69 Soluble protein extraction and enxyme assay 70 Glucan extrac tion and HPSEC analysis 72 Gel permeation chromatography (GPC) and high -performance anion exchange chromatography (HPAEC) analysis 74 Extraction of insoluble glucan in its native form 75 Microscopy 76 Sucrose gradient centrifugation 77 Results 77 Discussion 10 1 Acknowledgements 11 1 References 11 2 CHAPTER 4 : CONCLUSIONS AND RECOMMENDATIONS 117 Conclusions 117 Regarding M3 118 Regarding SM3 119 Additional observations 12 3 Recommendations for future experiments 12 4 (1) Further characterization of M3 & SM3 mutants 124 (2) Production of new mutants to determine need for glycogen Synthesis in Synechocystis sp. Strain PCC 6803 124 (3) Characterization of native enzymes involved in glycogen Metabolism in Synechocystis sp. Strain PCC 6803 125 (4) In vitro experimentation with glycogen synthases 125 (5) Ex pression of soluble starch synthases in combination with MSBE I 125 References 126 v ACKNOWLEDGEMENTS This is where I get t he privile ge of thanking all those who have helped me through the many years it has taken to complete this dissertat ion. I am very grateful to Professor Martin Spalding for giving me the opportunity to work in his lab and for tirelessly and patiently guiding me t hrough to the finish line. Professor Spalding had a keen understanding of what was needed to complete the job and kept me focused o n carrying out the necessary experiments and writing. Eve n with a busy schedule he has always been available to guide and direct my work and I am very grateful to him. Prof essor Jay -lin Jane has been a constant source of encouragement and a we alth of knowledge that I could refer to in working on this project and I owe her a debt of thanks. Prof essor David Oliver, Prof essor Paul Scott and Prof essor Madan Bhattacharyya my POS committee members and Prof essor Hanping Guan a former -POS committee mem ber , have been very helpful in giving of their time and expertise. I would also like to thank Prof essor Harry Horner and Tracey Pepper at the Bessey Microscopy Facility. They have assisted us in carrying out microscopic analyses of our samples and have b een an immense source of help. I am very grateful to them. Those who worked with me in the Spalding Lab as graduate students , Kyujung Van, Yingjun Wang, Parijat Juvale, Duanmu Dequiang, Andrea Rouse, Peter Vance and Wei Her and as post -doctoral student s, Drs. Ryan Wagner and Yoshiko Nakamura , have all enriched my time here at Iowa State University in many different ways. The undergraduate students Jake Sockness and Chris Coghlan have helped me a great deal in makin g solutions, pouring plates etc. , especial ly when I was pregnant with my two little girls, Anag i and Amila , and I appreciate them ve ry much. The post -doctoral students in Professor Jane s’ lab, Dr s. Dongsoon Suh and Zihua Ao taught me how to operate various instruments and Dr. Sathaporn Srichuwong taught me to make ‘pretty picture s’ of my data . I am also thankful to Li Li who worked on a si milar project as mine and Jovin Hasjim in Professor Jane s’ lab for their help on different occasions. My husband, Shantha Pieris , and my pa rents have invested of themselves to help me graduate and I would not have completed my work if not for their prayers, love , and timely help. vi Shantha has been my constant friend and helper through out. My sister and my br other have encouraged me through out . My two daughters , A nagi and Amila , have been a powerful motivation to help me complete my research . My extended family has also been a loving support to me . The task of trying to u nderstand, at least in part, a process that may impact granule formation has b een challenging and rewarding. I am filled with thankfulness and gratitude to the Creator of the Starch granule, Jesus Christ, for sustaining me through the experi ments and the many years of toil that have resulted in this dissertation. How can I completely and fully pen my gratitude to Him? My hope and str ength is ultimately founded in Him alone. Everything else pales into insignificance when compared to Him. He holds the key to all knowledge and is graciously allowing us to dis cover the secrets of His creation. What a wo nderful and merciful Creator God He is! vii PREFACE Since this dissertation is a scientific report of what was done and what results were obtained it does not portray, to any extent, my thoughts regarding this research and the processes that I have worked through in completing this research. Therefore, I wanted to take a few minutes to write down some of these things so that if some one , at some point in time , some where in the world does read this dissertation they will come to know more than scien ce and be directed towards Jesus Christ, who is the Creator God of all that is good and who is the author of life and in whom we can have the gift of eternal life. I am one who has accepted the redemption that Jesus alone can offer to us by His death and resurrection and am one who strives to allow Him to be the LORD of my life. The primary reason that propelled me to enroll in a Ph.D. program in Plant Physiology was to see how God through Jesus Christ has intricately and beautifully created all things wit h so much order and purpose. (It is interesting to me how the scientific community talks about the ‘structure function relationship’ and sometimes, if not always, misses the point that it is God who has created all things , including man, with a plan and a purpose! Even children have an instinct that says all thing s have a plan and purpose and do not exist because of random chance which is why they probably ask , at every opportunity , the question ‘Why?’.
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