MASTERARBEIT Titel der Masterarbeit "Construction of different mutants of Natrialba magadii and the influence of different φCh1 ORFs on N. magadii" verfasst von Léa Schöner, Bsc angestrebter akademischer Grad Master of Science (MSc) Wien, 2013 Studienkennzahl lt. Studienblatt: A 066 830 Studienrichtung lt. Studienblatt: Masterstudium Molekulare Mikrobiologie und Immunbiologie Betreut von: Ao. Univ.-Prof. Dipl.-Biol. Dr. Angela Witte 2 3 4 Table of Contents 1. Introduction ........................................................................................................................... 9 1.1. Archaea .............................................................................................................................. 9 1.1.1. Classification of the living world and the third domain of life ............................................ 9 1.1.2. Diversity of Archaea and the two major phyla - An overview .......................................... 10 1.1.3. Unique features of Archaea .............................................................................................. 12 1.1.3.1. Ether-linked lipids - an archaeal signature ................................................................ 13 1.1.3.2. Diversity of archaeal cell walls ................................................................................... 14 1.1.4. Archaea compared to the other 2 domains ...................................................................... 15 1.1.4.1. Transcription and Translation .................................................................................... 15 1.1.4.2. Replication ................................................................................................................. 16 1.1.4.3. Chromatin .................................................................................................................. 17 1.1.4.4. Polyploidy .................................................................................................................. 18 1.1.5. Halophilic and haloalkaliphilic Archaea ............................................................................ 18 1.1.5.1. Adaption to high salt concentrations - osmotic balance ........................................... 19 1.1.5.2. Lipids and Membranes .............................................................................................. 20 1.1.5.3. Halophilic proteins ..................................................................................................... 20 1.1.5.4. Haloalkaliphiles and adaption to high pH .................................................................. 21 1.1.5.5. Extracellular proteases and proteolytical growth ...................................................... 22 1.1.5.6. Lake Magadi - an alkaline soda lake .......................................................................... 22 1.1.6. Natrialba magadii ............................................................................................................. 23 1.1.6.1. Transformation of Natrialba magadii ........................................................................ 24 1.1.6.2. Shuttle vectors and selectable markers ..................................................................... 25 1.1.7. Promoters of halophilic Archaea ...................................................................................... 25 1.2. Viruses of Archaea ............................................................................................................ 27 1.2.1. From bacterial viruses to viruses infecting Archaea - An overview .................................. 27 1.2.2. Viruses of the Euryarchaeota ............................................................................................ 28 1.2.3. Viruses of the Crenarchaeota ........................................................................................... 29 1.2.4. Haloarchaeal viruses ......................................................................................................... 30 1.2.5. φCh1 .................................................................................................................................. 31 1.2.5.1. General characteristics .............................................................................................. 32 1.2.5.2. Organization of the genome and sequence features ................................................ 33 1.2.5.3. Gene regulation of lysogenic state and the lytic life cycle ......................................... 35 5 2. Material and Methods .......................................................................................................... 38 2.1. Material ............................................................................................................................ 38 2.1.1. Bacterial and Archaeal strains ........................................................................................... 38 2.1.2. Media ................................................................................................................................ 38 2.1.3. Antibiotics and other additives ......................................................................................... 40 2.1.3.1. E.coli ........................................................................................................................... 40 2.1.3.2. Natrialba magadii ...................................................................................................... 40 2.1.4. Restriction Enzymes, DNA-Polymerases and other DNA-modifying Enzymes .................. 41 2.1.5. Marker .............................................................................................................................. 42 2.1.5.1. DNA ladders ............................................................................................................... 42 2.1.5.2. Protein ladders .......................................................................................................... 42 2.1.6. Buffers and Solutions ........................................................................................................ 43 2.1.6.1. DNA gel electrophoresis ............................................................................................ 43 2.1.6.2. SDS PAGE and Western blot ...................................................................................... 43 2.1.6.3. Antibodies .................................................................................................................. 44 2.1.6.4. Protein purification under denaturing conditions ..................................................... 45 2.1.6.5. Southern blot ............................................................................................................. 45 2.1.6.6. Competent cells - E. coli (XL1-Blue, Rosetta) ............................................................. 46 2.1.6.7. Competent cells and transformation of Nab. magadii .............................................. 46 2.1.6.8. Isolation of chromosomal DNA of Nab. magadii ....................................................... 47 2.1.6.9. Isolation of φCh1 virus particles ................................................................................. 47 2.1.7. Plasmids ............................................................................................................................ 48 2.1.8. Primers .............................................................................................................................. 49 2.2. Methodes .......................................................................................................................... 50 2.2.1. DNA electrophoresis ......................................................................................................... 50 2.2.1.1. Agarose gel ................................................................................................................ 50 2.2.1.2. 6% Polyacrylamid gel ................................................................................................. 50 2.2.1.3. Staining of DNA .......................................................................................................... 50 2.2.2. Polymerase chain reaction ................................................................................................ 50 2.2.3. DNA purification ................................................................................................................ 52 2.2.3.1. Purfication of DNA samples ....................................................................................... 52 2.2.3.2. Gel elution ................................................................................................................. 53 2.2.4. Restriction ......................................................................................................................... 53 2.2.5. DNA modifications ............................................................................................................ 53 6 2.2.5.1. Fill-in 5' overhangs ..................................................................................................... 53 2.2.5.2. Dephosphorylation .................................................................................................... 54 2.2.5.3. Ligation ...................................................................................................................... 54 2.2.6. Transformation of E. coli ................................................................................................... 54 2.2.6.1.