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Generation of Lipid-Rich Mutants And GENERATION OF LIPID-RICH MUTANTS AND CHARACTERIZATIONS IN GREEN ALGA MICROALGA DUNALIELLA TERTIOLECTA LINA YAO (B.Sc. (Hons.), Zhejiang University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF MICROBIOLOGY AND IMMUNOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2016 Declaration Declaration I hereby declare that the thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. _________________ Lina Yao (18 July 2016) i Acknowledgements My utmost and sincere thanks to: My supervisor, A/P Lee Yuan Kun, for his superb guidance throughout my PhD years. Thank you for providing me with this opportunity to work in this green lab. You supervised me in my research direction patiently, and encouraged me by generously sharing your own experience. Your grace, vitality, and passion for work inspired me a lot. As a newbie who just stepped into the research gate and independent life, I am extremely grateful to you for all you did to enhance my experience here. Thank you for sparing a lot of your busy time helping me through the emails or my unexpected visits. A/P Tan Tin Wee, for co-supervising me in the bioinformatics. Very special thanks to you for igniting the spark of the powerful programming languages in my head three years ago. It was from you, I realized how important to apply this technology into conventional molecular biological research. And it was from you, I found out enthusiasms and creativities could really motivate a scientist in his/ her research. Your sincere attitude towards work inspired me a lot for my future career. I would also like to express my thanks to Assistant Professor Ban Hon Kim Kenneth, who also guided me in the programming skills. The great people I met and wonderful experiences will help me to continue understanding the multidisciplinary science in the future. Our lab officer, Mr Low Chin Seng, for his impressive management to make the lab run in order so that we could focus on our own research. And also for his good humor and generousness to us juniors, which brought many joys ii and fun during these years. From him, I learnt many skills to solve small technical problems in lab creatively. To our previous research assistant Ms Lin Hui Xin (current PhD student) for helping me to begin with my research from small tiny lab techniques and supporting me all along with the Nile red quantitative work. Without her, I would have a hard time struggling with the trials and errors by myself. It is Mr Low and Hui Xin who introduced the colorful life of Singapore to me when I came here for the first time. I would also like to express my thanks to our research assistant Mr Ezekiel Ho Hee Zhern (previous), Mr Chong Si Cong (previous), laboratory technologist Ms Voong Miao Lian for their great help in supporting the lab work and consumables. Without those people, all things would descend into a mass. Senior post-doctoral research fellow, Dr Shen Hui, who spent a lot of time guiding me in molecular biology in details by her expertise, and always encouraged me when I felt down by her own experience. Dr Shen also did a lot to create a lovely lab environment for us to feel easy and warm. I also want to express my thanks to Dr Ng Yi Kai, post-doctoral research fellow, who not only guided us in the techniques and supported us by creating the general platforms of next-generation sequencing, total fatty acid measurement and metabolite profiling, but also played with us and brought us a lot of laughs and entertainments brotherly. Thanks to Dr Ng, for his kind help to read and check this thesis and provided me with great suggestions. And to all my lab colleagues, post-graduate students, Dr Zhao Ran (graduated), Dr Ng Hui Ping Daphne (graduated), Koh Ting Wei Kelvin, Tan Wei Min Kenneth, Siti Radiah Binte Safie, post-doctoral research fellow Dr Junhui Huang, visiting Assistant Professor Jin Liu and exchange students iii Xiaonian Ma, Zhao Zhang and Dongzhe Sun from PKU, for all their help, time spent together, and good cheer. To our collaborator, Professor Liang Li’s group from University of Alberta, for providing us with great support in our metabolomic study. As always, first and foremost, my extraordinary parents and family members, for their endless support and encouragement. My boyfriend, Yuan, for his accompany during these years and technical support in my research using his expertise in computer science. We shared our happiness and frustration in our PhD life together, and brainstormed using our multidisciplinary knowledge. I would also like to express my gratitude to Department of Microbiology and Immunology, National Research Foundation (NRF) under its Campus for Research Excellence and Technological Enterprise (CREATE) Programme, and the NUS Research Scholarship to support me for this work. Lastly, to all the specialists, seniors, friends, administration staffs who I met in my PhD life that I did not mention in this brief list, I am eternally grateful for all you did to enhance my PhD life physically and mentally. iv TABLE OF CONTENTS Declaration………………………………………………………….……..….i Acknowledgements…………………………………………………..………ii TABLE OF CONTENTS…………………………………………….………v Summary………………………………………...……………..…………...xii List of Tables……………………………………………………………….xiv List of Figures………………………………...……….……...……………xv List of Abbreviation…………………………..……….....…….…............xviii Chapter 1 Introduction and literature review…………...…….……..............1 1.1 Fossil fuel crises and prospect of microalgae as biofuel…………..……..1 1.2 Lipid accumulation in microalgae………………………………………..2 1.3 Biosynthesis of lipid droplets……………………………….………........3 1.4 Numerous approaches to increase triacylglycerol (TAG) productivity….4 1.4.1 Genetic engineering approach…………………………………..……...5 1.4.2 Biochemical approach………………………………………..………...5 1.5 Organism of interest: Dunaliella tertiolecta………………………..........6 1.6 Research gap………………………………………….……………..........7 1.6.1 Rapid TAG measurement method………………………....…………...7 1.6.2 D. tertiolecta transcriptome database…………………………………..7 v 1.6.3 Understanding of regulatory ………………………………….………..8 1.6.4 Cell growth and lipid accumulation…………........................................9 1.7 Project objectives and hypotheses……………………..………..............10 1.8 Significance and scope of this study………………………………........11 Chapter 2 Materials and methods………………...……........…...…...........12 2.1 Culture conditions……………………………………………….……...12 2.2 RNA extraction……………...…………………………………………..13 2.3 DNA extraction……………………………………………………........13 2.4 Determination of photosynthetic rate and photosynthetic efficiency…...14 2.5 Determination of glycerol and starch content………………….……….14 2.6 Cloning of D. tertiolecta gene………………………...……..………….14 2.7 Analysis of D. tertiolecta gene …………………………………………15 2.8 D. tertiolecta transformation……………………………………............16 2.8.1 Plasmid construction………………………………………………….16 2.8.2 D. tertiolecta transformation……………………………………….....16 2.8.2.1 Via glass-bead method………………………….…………….…….16 2.8.2.2 Via electroporation method………………………….…….………..17 2.8.3 Mutant screening and selection………………………….….……...…17 Chapter 3 Optimization of quantitative measurement of neutral lipids using Nile red staining………………...………………………………..…………18 vi 3.1 Introduction………………………………………….………………….18 3.2 Materials and Methods………………………………………………….18 3.2.1 Culture and reagent preparation for Nile red staining…………..…….18 3.2.2 Optimization of instrumental parameters…………………...………...20 3.2.3 Application on nitrogen deprivation (ND) study………….....……….20 3.2.4 Gas chromatography mass spectrometry (GC-MS) for total FA measurement………………………………………………………………...20 3.3 Results and Discussion……………………………...……………..……21 3.3.1 Optimizing instrument parameters and staining conditions in Nile red method…………………………………………………...………….............21 3.3.2 Comparing the direct staining method and standard addition method..23 3.3.3 Neutral lipid is triggered by ND ……………………………………...25 3.3.4 Visualization of neutral lipid droplet through fluorescence microscopy………………………………………………………………….27 3.3.5 Comparison of Nile red assay with GC-MS measurement….………..27 3.4 Conclusion………………………….…………………………………...29 Chapter 4 Generation of lipid-rich mutant……………………….………...30 4.1 Introduction…………………………………………..…………………30 4.2 Materials and Methods………………………………………………….30 4.2.1 Culture strains and physiological study……………………………….30 vii 4.2.2 Generation of D. tertiolecta mutants………………………………….30 4.2.2.1 Mutant with overall increased TAG production…………………….31 4.2.2.2 Mutant with TAG accumulation in exponential growth phase (TAEP)……………………………………………………………………...31 4.3 Results and Discussion…………………………………………….........33 4.3.1 Screening and selection of lipid-rich mutants …………………….….33 4.3.2 Physiological studies for the D9 mutant………………………………34 4.3.3 FACS enriched a pool of mutant strains and generation of G11_7…...36 4.4 Conclusion………………………………………………………………44 Chapter 5 Construction of Bag2D package and its application in characterizing D9 mutant...............................................................................45 5.1 Introduction……………………………………………………………..45 5.2 Materials and Methods………………………………………………….45 5.2.1 Construction of cDNA libraries for NGS analysis……………….…...45 5.2.2 De novo assembly of Illumina short reads and RNA-Seq data processing…………………………………………………………….……..46 5.2.3 Comparisons among other green algae and high plant species…….....47
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