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Developing a Tool to Characterize the Ultradian Rhythm in Diploid Louisiana State University LSU Digital Commons LSU Master's Theses Graduate School 2011 Developing a tool to characterize the ultradian rhythm in diploid Saccharomyces cervisiae using the reporter gene green fluorescent protein Imran Chiragh Louisiana State University and Agricultural and Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_theses Part of the Chemical Engineering Commons Recommended Citation Chiragh, Imran, "Developing a tool to characterize the ultradian rhythm in diploid Saccharomyces cervisiae using the reporter gene green fluorescent protein" (2011). LSU Master's Theses. 530. https://digitalcommons.lsu.edu/gradschool_theses/530 This Thesis is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Master's Theses by an authorized graduate school editor of LSU Digital Commons. For more information, please contact [email protected]. DEVELOPING A TOOL TO CHARACTERIZE THE ULTRADIAN RHYTHM IN DIPLOID SACCHAROMYCES CEREVISIAE USING THE REPORTER GENE GREEN FLUORESCENT PROTEIN A Thesis Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Master of Science in Chemical Engineering in The Gordon A. and Mary Cain Department of Chemical Engineering by Imran Chiragh B.S. University of New Mexico, Albuquerque, NM 2007 May 2011 DEDICATION To my incredible parents Dr. Abdul-Latif Chiragh and Wajahat-un-Nisa Chiragh who have and continue to inspire, enlighten, and motivate me everyday ii ACKNOWLEDGMENTS I would like to begin by thanking Allah (SWT) for bestowing upon me the guidance and ability to seek knowledge and pursue a masters‟ degree in the challenging field of chemical engineering. I would like to thank Dr. Michael Benton, my advisor and thesis chair, for his support and encouragement. His expertise and guidance has made my endeavor enlightening and will remain with me as I continue my career. I would also like to thank my committee members Dr. James Henry and Dr. Martin Hjortsø for their valuable recommendations pertaining to this study and assistance in my professional development. I would like to thank Rong Bai for his guidance in the laboratory, his eagerness to see me succeed, and most importantly his friendship. His ability and expertise made my thesis work possible. I would like to thank John Tate for reviewing and editing my thesis. His expertise allowed me to properly convey my work. I would like to thank my group members William Barrett Ainsworth, Murali Koneru, Courtney Lane, and Zenghui Zhang for their help in the laboratory and understanding of the field. I would like to thank my classmates Oladapo Ayeni, Rohit Ghosh, Edgardo Kamar, Tejaswini Narayana, Khue Nyugen, and Gregory Robertson. I would like to thank Kimberly LeBlanc from Department of Biological Sciences for her guidance in DNA extraction in bacteria and yeast. Also, I would like to thank Marilyn Dietrich at the School of Veterinary Medicine, LSU for support and assistance in flow cytometry. iii I would like thank my roommate Yosef Javed and my good friends Ammar Qureshi and Mohammad Imran Chaudhry. I would like to thank my sister and brother, Atifa Chiragh and Furqan Chiragh. I am always inspired by their love, compassion, support, and faith in me. I would love to thank my parents, Dr. Abdul-Latif Chiragh and Wajahat-un-Nisa Chiragh. Without their sacrifice and resilience I would not have had the opportunities to enhance my education and achieve what I have. Because of their unconditional love and support I am here. I can never repay or thank them enough for all what they have done for me and continue to do. I will always love and cherish you, Abuji and Amiji! iv TABLE OF CONTENTS DEDICATION ......................................................................................................................................ii ACKNOWLEDGMENTS.................................................................................................................. iii LIST OF TABLES..............................................................................................................................vii LIST OF FIGURES .......................................................................................................................... viii ABSTRACT .......................................................................................................................................... x CHAPTER 1 – INTRODUCTION ...................................................................................................... 1 1.1 Motivation and Objective ................................................................................................... 1 1.2 History of Biological Rhythms........................................................................................... 4 1.3 Criteria of a Biological Rhythm ........................................................................................ 7 1.4 Summary ............................................................................................................................. 10 CHAPTER 2 – LITERATURE REVIEW OF THE CIRCADIAN RHYTHM.............................. 12 2.1 Disease and Disease Progression Related to the Circadian Rhythm ......................... 12 2.2 Circadian Rhythm Sleep Disorder.................................................................................. 12 2.3 Smith-Magenis Syndrome ................................................................................................ 16 2.4 Disrupted Circadian Effects on Testosterone ............................................................... 19 2.5 Circadian Rhythm’s Effect on Alzheimer’s Disease .................................................... 20 2.6 Summary ............................................................................................................................. 22 CHAPTER 3 – LINK BETWEEN THE CIRCADIAN AND ULTRADIAN SYSTEMS ............ 24 3.1 Introduction ........................................................................................................................ 24 3.2 Ultradian Rhythm in Yeast .............................................................................................. 26 3.3 Yeast Glycolysis ................................................................................................................. 28 3.4 Gene Expression in Yeast ................................................................................................. 30 3.5 Gene Selection .................................................................................................................... 32 3.6 Synchronization of a Large Yeast Cell Population ...................................................... 36 3.6.1 Induction Methods......................................................................................................... 37 3.6.2 Centrifugal Elutriation ................................................................................................. 41 3.6.3 Summary ......................................................................................................................... 42 CHAPTER 4 – MATERIALS AND METHODS ............................................................................ 44 4.1 Introduction to Materials and Methods ......................................................................... 44 4.2 Yeast Medium .................................................................................................................... 44 4.3 Creation and Verification of Deleted Ultradian Genes ............................................... 44 4.4 Synchronization of Yeast Cell Populations ................................................................... 46 4.5 Flow Cytometry Experiments of Asynchronous and Synchronous Populations .... 48 v CHAPTER 5 – RESULTS AND DISCUSSION.............................................................................. 51 5.1 Transformed Yeast Strain ICY 100................................................................................ 51 5.2 Transformed Yeast Strain ICY 101................................................................................ 55 5.4 Synchronization of a Largest Yeast Population Using Block-and-Release .............. 59 5.5 Analyzing GFP from Synchronized ICY 100 in Batch ................................................ 62 5.6 Analyzing GFP from Synchronized ICY 100 in Continuous Flow ............................ 66 CHAPTER 6 – CONCLUSION AND FUTURE WORKS ............................................................. 70 REFERENCES ................................................................................................................................... 72 VITA.................................................................................................................................................... 79 vi LIST OF TABLES Table 1: PCR primers used to construct knockout cassette ............................................................. 45 Table 2: Primers constructed to verify transformation ..................................................................... 46 Table 3: HU 200mM ICY 100 Culture Cell Cycle Percentages over Time with Standard Deviation. ............................................................................................................................................ 60 Table 4: NOCO 15 μg/mL ICY 100 Culture Percentages of Budded
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