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Masterarbeit / Master's Thesis MASTERARBEIT / MASTER’S THESIS Titel der Masterarbeit / Title of the Master‘s Thesis „ Establishing a Workflow for Exploring the Diversity and Environmental Distribution of Chlamydiae “ verfasst von / submitted by Tamara Halter BSc angestrebter akademischer Grad / in partial fulfilment of the requirements for the degree of Master of Science (MSc) Wien, 2016 / Vienna 2016 Studienkennzahl lt. Studienblatt / A 066 830 degree programme code as it appears on the student record sheet: Studienrichtung lt. Studienblatt / Masterstudium Molekulare Mikrobiologie, Mikrobielle degree programme as it appears on Ökologie und Immunbiologie the student record sheet: Betreut von / Supervisor: Univ.-Prof. Dr. Matthias Horn “We can only see a short distance ahead, but we can see plenty there that needs to be done.” ― Alan Turing ACKNOWLEDGEMENTS First of all, I would like to thank Michael Wagner for giving me the opportunity to work in his lab and Matthias Horn for welcoming me in his group. A special thanks goes to Astrid Horn for a really great supervision. Thank you for always being patient and encouraging me whenever I felt lost. Of course, I also would like to thank all DOMiES for the nice and familiar working atmosphere and especially the symbiosis group (Allen, Stephan, Paul, Flo W., Vincent, Cecilia, Jasmin, Lena, Gabi) for supporting me and all the nice and productive discussions. Further, I would particularly like to thank Stefano Fazi, Rok Kostanjsek, Julia Vierheilig and Petra Pjevac for providing samples for the project. Finally, I also would like to thank Nadia for a really great friendship and for all the fun we had in the lab and my mom and sister for their unconditional support. TABLE OF CONTENTS 1 INTRODUCTION ............................................................................................................................ 1 1.1 The phylum Chlamydiae ....................................................................................................... 1 1.2 Aims of the project ................................................................................................................ 3 2 MATERIAL AND METHODS ........................................................................................................... 5 2.1 Material ................................................................................................................................. 5 Chemicals ....................................................................................................................... 5 Consumables .................................................................................................................. 6 Media, buffers and solutions ........................................................................................ 6 Enzymes and Reagents .................................................................................................. 9 Kits ................................................................................................................................ 10 Organisms .................................................................................................................... 10 FISH probes .................................................................................................................. 10 PCR primers .................................................................................................................. 10 Technical Equipment ................................................................................................... 11 Software ....................................................................................................................... 12 2.2 Methods............................................................................................................................... 13 Biogeography of Chlamydiae ...................................................................................... 13 2.2.1.1 Primer design and evaluation .............................................................................. 13 2.2.1.2 MiSeq amplicon sequencing................................................................................ 14 2.2.1.3 DNA Isolation of Lago di Paola filters .................................................................. 17 2.2.1.4 MiSeq amplicon sequencing................................................................................ 17 2.2.1.5 Statistical and phylogenetic analysis ................................................................... 18 2.2.1.6 Adaptation and re-evaluation of PCR settings ................................................... 18 Microdiversity of Rhabdochlamydia porcellionis ....................................................... 20 2.2.2.1 Sampling and animals .......................................................................................... 20 2.2.2.2 Preparation of the animals and hepatopancreases ........................................... 20 2.2.2.3 Screening of woodlice populations ..................................................................... 20 2.2.2.4 Cultivation of Rhabdochlamydia porcellionis ..................................................... 21 2.2.2.5 Screening of Sf9 cultures ..................................................................................... 22 2.2.2.6 High molecular weight DNA isolation for genome sequencing ......................... 23 3 RESULTS ...................................................................................................................................... 24 3.1 Biogeography of Chlamydiae .............................................................................................. 24 DNA Isolation ............................................................................................................... 24 PCR and specificity of the PCR primers ...................................................................... 24 Evaluation of the mock communities ......................................................................... 25 Statistical analysis ........................................................................................................ 26 3.1.4.1 Community composition ..................................................................................... 26 3.1.4.2 Non-metric multidimensional scaling and perMANOVA ................................... 28 3.1.4.3 Similarity percentages analysis ........................................................................... 29 Phylogenetic analysis................................................................................................... 30 Re-evaluation of the PCR primers ............................................................................... 30 3.1.6.1 Specificity of the primers ..................................................................................... 30 3.1.6.2 Influence of increased cycle number .................................................................. 32 3.2 Microdiversity of Rhabdochlamydia porcellionis ............................................................... 34 Screening of woodlice populations ............................................................................ 34 Cultivation of Rhabdochlamydia porcellionis from Slovenian population ................ 35 High molecular weight DNA Isolation ......................................................................... 35 4 DISCUSSION ................................................................................................................................ 36 4.1 Biogeography of Chlamydiae .............................................................................................. 36 DNA Isolation and PCR ................................................................................................ 36 Evaluation of the mock communities ......................................................................... 36 Composition and dynamics of chlamydial communities ........................................... 37 Phylogenetic analysis................................................................................................... 38 Re-evaluation of the PCR primers ............................................................................... 39 Concluding remarks and outlook ................................................................................ 39 4.2 Microdiversity of Rhabdochlamydia porcellionis ............................................................... 40 Screening of woodlouse populations ......................................................................... 40 Cultivation of Rhabdochlamydia porcellionis ............................................................. 41 High molecular weight DNA isolation ......................................................................... 41 Concluding remarks and outlook ................................................................................ 41 5 LIST OF ABBERIVATIONS ............................................................................................................ 43 6 REFERENCES ............................................................................................................................... 45 7 ABSTRACT ................................................................................................................................... 49 8 ZUSAMMENFASSUNG ................................................................................................................ 50 9 SUPPLEMENT .............................................................................................................................
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