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Cell- and Genome Size Responses to Different Temperatures in Haptophytes

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Cell- and Genome Size Responses to Different Temperatures in Haptophytes Cell- and genome size responses to different temperatures in haptophytes A long-term experiment study on the algal species Prymnesium kappa and Calyptrosphaera sp Erik Standeren Master Thesis Molecular Biology University of Oslo (UiO) Department of Biosciences Section for Aquatic Biology and Toxicology (AQUA) UNIVERSITY OF OSLO [09 / 2018] II Acknowledgement All the work presented in this master thesis war conducted at the Section for Aquatic Biology and Toxicology (AQUA) at the Department of Biosciences, University of Oslo (UiO) from the period of 1st January, 2017 to 30th September, 2018. But before I start there is quite a few persons that I need to thank. First of all, I took my bachelor at the University of Bergen (UiB) and transferred to Oslo. Secondly, I took my bachelor in Molecular Biology, and when this project is just as much Marine biology, there is a lot of new techniques that I have done throughout this period of work, that I have never been close to doing. There is therefore a lot of people I have to thank. All from friendly students to professors. All the students at Molecular Biology I have met have been really nice and welcoming. There was never a moment when I did not feel included. As I mentioned, there have been a lot of new techniques I had to learn and to help me with that, a lot of friendly professors, and other students and post-docs, never hesitated using some of their free time to give me a hand. The first one I have to give my thanks to is my supervisor, professor Dag Olav Hessen. He is one of the most recognised biologists in Norway, and that for a good reason. He always greets you with a smile and shows a great interest in his students work. He was maybe not the one that helped me the most in respect to laboratory work, but he surely made up with commitment and never hesitated to introduce me to other, both postdocs and professors, that could help me. His passion for his theory was the reason for why I chose this assignment and I never regretted that. My co-supervisor, prof. Bente Edvardsen, also deserve a huge thank. Her knowledge and experience, not only in Flow Cytometry, but also almost anything that had something with algae, has been to great help. Senior Engineer Sissel Brubak was an important person for me to get started. She showed how to create the medium I needed to grow my algae. And autoclaving is an important part of medium-making, and a lot of frustration has been produced with good help of the autoclave which more than not, had some kind of problem. Rita Amundsen, Engineer Roy Falleth and III again, Sissel Brubak, was all important for me, not only to get help when the autoclave did not work, but also to share my frustration. Rita Amundsen also deserve a special gratitude for maintaining and diluting my cultures while I was away on a two-week vacation. Maybe the person I had the most conversation with, was Aqua engineer, Per-Johan Færøvig. Flow Cytometry was the most used method of my thesis, and Færøvig was the one that helped me to become familiarized with the instrument. His “learning by doing” teaching method was really important for me to learn in a way that I actually learned something. I tried doing a lot on my own, but whenever I crashed into a problem-wall, he was quick to give me help and advice. He was also really helpful with the experimental setup and introduced me to the Casy Cell-counter. Dr. Marwa Jalal also deserve some of my gratitude for giving me some important, new insight to my Flow Cytometry preparation and technique. Senior Engineer Berit Kaasa also deserve my thanks for assisting me with the isolation of DNA and showing me the Qubit DNA concentration instrument and stoichiometry (C:N:P) analysis. Jon Bråte and Øyvind Gulbrandsen, and rest of the sequencing team, I thank you for your help with analysing of my data. Assoc. prof. Wenche Eikrem isolated the algae I used during my thesis and she also greatly assisted med to look at the algae in TEM. Postdoc Luka Supraha assisted me with visualizing my algae in SEM. Thank you, both. The last one I have to thank is postdoc Francisco José Bullejos Carrillo. He spent large amounts of his time unselfishly assisting me with Protein, RNA and C:N:P analysis, teaching me new techniques and helping me analyse the results. Erik Standeren Oslo, 30th September, 2018. IV © Erik Standeren 2018 Cell- and genome size responses to different temperatures in haptophytes Erik Standeren http://www.duo.uio.no/ Printed: Reprosentralen, University of Oslo V Table of Content Acknowledgement................................................................................................................... III ABSTRACT.......................................................................................................................... VIII 1. INTRODUCTION ................................................................................................................ 1 1.1 Temperature and Organism Size ................................................................................. 1 1.2 The Basics of Algae .................................................................................................... 3 1.3 Genome Size, in relationship with cell size, in eukaryote organisms …..................... 5 1.4 Flow Cytometry .......................................................................................................... 8 1.5 Goals and Hypothesis ................................................................................................ 10 2. MATHERIAL AND METHODS ....................................................................................... 12 2.1 Model Organisms ...................................................................................................... 12 2.2 Experimental Setup ................................................................................................... 14 2.3 Medium ..................................................................................................................... 17 2.4 Cell Number and Cell Size Measurement ................................................................. 17 2.4.1 Light Microscopy Measurement ............................................................................ 18 2.4.2 Casy Cell Counter Measurement ........................................................................... 19 2.5 Genome Size Estimation Using Flow Cytometry ..................................................... 20 2.5.1 Cell Lysis ............................................................................................................... 21 2.5.2 Percoll Filtration .................................................................................................... 23 2.5.3 Flow Cytometry – FCM ......................................................................................... 23 2.6 DNA Isolation and DNA Sequencing ....................................................................... 24 2.7 Electron Microscopy ................................................................................................. 25 2.7.1 Transmission Electron Microscopy (TEM)............................................................ 25 2.7.2 Scanning Electron Microscopy (SEM)................................................................... 26 2.8 RNA, Protein and C:N:P analysis ............................................................................. 26 2.8.1 RNA Quantifying Analysis .................................................................................... 27 2.8.2 Protein Quantifying Analysis ................................................................................. 28 2.8.3 C:N:P Ratio Analysis ............................................................................................. 29 3. RESULTS ........................................................................................................................... 30 3.1 Cell- and Genome Sizes Correlation Across Different Algal Species ….................. 30 3.2 Long-Term Temperature Experiment ....................................................................... 35 3.2.1 Cells Sizes and Cell Concentrations ...................................................................... 35 VI 3.2.2 Flow Cytometry ..................................................................................................... 39 3.2.3 Second FCM Results for Prymnesium kappa ........................................................ 45 3.2.4 Cell Size and Genome Size Correlation ................................................................. 50 3.3 Electron Microscopy ................................................................................................. 52 3.3.1 Transmission Electron Microscopy ....................................................................... 52 3.3.2 Scanning Electron Microscopy ............................................................................. 54 3.4 C:N:P, RNA and Protein Analysis ............................................................................ 55 3.4.1 RNA Quantifying Analysis ................................................................................... 55 3.4.2 Protein Quantifying Analysis ................................................................................ 57 3.4.3 C:N:P Ratio Analysis ............................................................................................. 59 3.5 DNA Sequencing ....................................................................................................... 60 3.5.1 DNA Sequencing – K-mer
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