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Prokaryotic Biodiversity of Lonar Meteorite Crater Soda Lake Sediment and Community Dynamics During Microenvironmental Ph Homeostasis by Metagenomics

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Prokaryotic Biodiversity of Lonar Meteorite Crater Soda Lake Sediment and Community Dynamics During Microenvironmental Ph Homeostasis by Metagenomics Prokaryotic Biodiversity of Lonar Meteorite Crater Soda Lake Sediment and Community Dynamics During Microenvironmental pH Homeostasis by Metagenomics Dissertation for the award of the degree "Doctor of Philosophy" Ph.D. Division of Mathematics and Natural Sciences of the Georg-August-Universität Göttingen within the doctoral program in Biology of the Georg-August University School of Science (GAUSS) Submitted by Soumya Biswas from Ranchi (India) Göttingen, 2016 Thesis Committee Prof. Dr. Rolf Daniel Department of Genomic and Applied Microbiology, Institute of Microbiology and Genetics, Faculty of Biology and Psychology, Georg-August-Universität Göttingen, Germany PD Dr. Michael Hoppert Department of General Microbiology, Institute of Microbiology and Genetics, Faculty of Biology and Psychology, Georg-August-Universität Göttingen, Germany Members of the Examination Board Reviewer: Prof. Dr. Rolf Daniel, Department of Genomic and Applied Microbiology, Institute of Microbiology and Genetics, Faculty of Biology and Psychology, Georg-August-Universität Göttingen, Germany Second Reviewer: PD Dr. Michael Hoppert, Department of General Microbiology, Institute of Microbiology and Genetics, Faculty of Biology and Psychology, Georg-August-Universität Göttingen, Germany Further members of the Examination Board: Prof. Dr. Burkhard Morgenstern, Department of Bioinformatics, Institute of Microbiology and Genetics, Faculty of Biology and Psychology, Georg-August-Universität Göttingen, Germany PD Dr. Fabian Commichau, Department of General Microbiology, Institute of Microbiology and Genetics, Faculty of Biology and Psychology, Georg-August-Universität Göttingen, Germany Prof. Dr. Kai Heimel, Department of Molecular Microbiology and Genetics, Institute of Microbiology and Genetics, Faculty of Biology and Psychology, Georg-August-Universität Göttingen, Germany PD Dr. Wilfried Kramer, Department of Molecular Genetics, Institute of Microbiology and Genetics, Faculty of Biology and Psychology, Georg-August-Universität Göttingen, Germany Date of the oral examination: 04.08.2016 “A man should look for what is, and not for what he thinks should be.” - Albert Einstein Dedicated to my family and friends. TABLE OF CONTENTS Abbreviations ....................................................................................................................... 1 List of Figures ....................................................................................................................... 3 List of Tables ........................................................................................................................ 6 Summary .............................................................................................................................. 8 1 Introduction ............................................................................................................... 12 1.1 State Of The Research On Prokaryotes Inhabiting Soda Lakes.......................... 12 1.1.1 Prokaryotes Isolated From Soda Lakes ........................................................ 14 1.1.1.1 Prokaryotic Biodiversity And Biogeochemical Cycles In Soda Lakes .... 15 1.2 Soda Lake Environment ..................................................................................... 18 1.2.1 Origin Of Soda Lakes .................................................................................... 21 1.2.2 pH As A Major Environmental Variable ....................................................... 21 1.3 Element Cycling In The Soda Lake Environment ................................................ 22 1.3.1.1 Carbon Cycle ......................................................................................... 22 1.3.1.2 Nitrogen Cycle ...................................................................................... 26 1.3.1.3 Sulfur Cycle ........................................................................................... 27 1.4 Unique Nature Of Lonar ..................................................................................... 28 1.5 Biodiversity And Ribosomal RNA Genes ............................................................ 30 1.6 Community Dynamics And Microenvironmental ph Homeostasis.................... 31 1.7 Metagenomics ................................................................................................... 31 2 Objective And Importance ......................................................................................... 34 2.1 Objectives Of The Investigation ......................................................................... 34 2.2 Importance Of This Investigation ....................................................................... 35 3 Materials And Methods ............................................................................................. 38 3.1 Sediment Samples .............................................................................................. 38 3.2 Handling Of Equipment And Reagents .............................................................. 38 3.3 Media, Media Supplements And Antibiotics ..................................................... 38 3.4 Organisms, Oligonucleotides, Enzymes, And Buffers ........................................ 41 3.4.1 Strain Description......................................................................................... 41 3.4.2 Description Of Primers ................................................................................. 42 3.4.2.1 Primers For Roche Platform Sequencing .............................................. 42 3.4.2.2 Primers For Illumina Platform Sequencing ........................................... 42 3.4.3 Vectors ......................................................................................................... 43 3.4.4 Details Of The Enzymes ................................................................................ 46 3.4.5 Composition Of Buffers And Stock Solutions ............................................... 46 3.5 Commercial Kits ................................................................................................. 49 3.6 Computational And Statistical Analysis Tools .................................................... 50 3.7 DNA And RNA Extraction Methods .................................................................... 51 3.7.1 Extraction Of DNA And RNA For The Total And The Active Prokaryotic Biodiversity Assessment By Illumina Platform .......................................................... 51 3.7.2 Extraction Of DNA For Additional Biodiversity Assessment By Roche Platform 52 3.7.3 Extraction Of DNA From Agarose Gel .......................................................... 53 3.7.4 Extraction Of DNA For Direct Metagenome Analysis .................................. 54 3.7.5 Extraction Of DNA For The Study Of Community Dynamics During Microenvironmental pH Homeostasis ....................................................................... 54 3.7.6 Extraction Of DNA For Construction Of Metagenomic Libraries ................. 55 3.7.7 Extraction Of Plasmid DNA .......................................................................... 55 3.7.7.1 Isolation Of Plasmid Using Peqgold Plasmid Miniprep Kit ................... 55 3.7.7.2 Isolation Of Plasmid Using QIAGEN Plasmid Plus Midi Kit ................... 56 3.8 Quantification Of DNA ....................................................................................... 57 3.8.1 Determination Of DNA Concentration By Nano-Drop Spectrophotometer 57 3.8.2 Determination Of DNA Concentration By Qubit Fluorometer .................... 58 3.9 Construction And Screening Of Metagenomic Libraries ................................... 58 3.9.1 Enrichment Cultures .................................................................................... 58 3.9.2 Construction Of Small Insert Library ............................................................ 59 3.9.2.1 Preparation Of Electrocompetent Cells ................................................ 59 3.9.2.2 Small Insert Library ............................................................................... 59 3.9.2.2.1 Transformation Of E. Coli TOP10 And E. Coli DH5α Cells By Electroporation .................................................................................................. 60 3.9.2.2.2 Detection Of Recombinant Cells By Blue-White Screening ............. 61 3.9.2.2.3 Library Preparation .......................................................................... 61 3.9.2.3 Screening Of Small Insert Libraries ....................................................... 62 3.9.2.3.1 Protease Activity Screening ............................................................. 62 3.9.2.3.2 Lipase Activity Screening .................................................................. 62 3.9.3 Large Insert Library ...................................................................................... 62 3.9.3.1 Preparation Of Infection Cells .............................................................. 62 3.9.3.2 Shearing The Insert DNA ....................................................................... 63 3.9.3.3 End-Repair Of The Sheared DNA .......................................................... 63 3.9.3.4 Ligation Reaction .................................................................................. 63 3.9.3.5 Packaging Of Fosmid Clones ................................................................. 63 3.9.3.6 Infection ...............................................................................................
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