Characterisation of the CRISPR/Cas system of the hyperthermophilic Archaeum Thermoproteus tenax [Elektronische Ressource] / vorgelegt von André Plagens

Besitzer

Sujets

• Biologie

Informations

• Publié par: universitat_duisburg-essen
• Publié le: 01 janvier 2010
• Nombre de lectures: 29
• Langue: Deutsch
• Poids de l'ouvrage: 9 Mo

Extrait

Characterisation of the CRISPR/Cas system
of the hyperthermophilic Archaeum
Thermoproteus tenax



Inaugural-Dissertation

zur
Erlangung des Doktorgrades
Dr. rer. nat.
der Fakultät für
Biologie und Geografie
an der


Universität Duisburg-Essen


vorgelegt von
ANDRÉ PLAGENS
aus Bottrop




Februar 2010
Die der vorliegenden Arbeit zugrunde liegenden Experimente wurden am Institut für
Biologie in der Abteilung für Mikrobiologie I der Universität Duisburg-Essen, Campus
Essen durchgeführt.

1. Gutachter: Prof. Dr. Reinhard Hensel (Essen)
2. Gutachter: Prof. Dr. Hemmo Meyer (Essen)
3. Gutachter: Prof. Dr. Reinhard Sterner (Regensburg)
Vorsitzende des Prüfungsausschusses: Prof. Dr. Shirley Knauer (Essen)


Tag der mündlichen Prüfung: 23.06.2010
















































Man will nicht nur glücklich sein,
sondern glücklicher als die anderen.
Und das ist deshalb so schwer,
weil wir die anderen für glücklicher halten,
als sie sind.

Charles-Louis de Montesquieu Table of contents I
TABLE OF CONTENTS

LIST OF FIGURES .................................................................................................. V

LIST OF TABLES .. VI

1 INTRODUCTION ...................................................................................................... 1

2 MATERIAL AND METHODS ................................................................................... 9
2.1 Chemicals, enzymes, kits and consumables ................. 9
2.2 Instruments ..................................................................................................... 10
2.3 Strains and growth conditions ...... 12
2.3.1 Culture conditions for growing T. tenax ...................................................... 12
2.3.1.1 Culture conditions at increased ionic strength ................................ 12
2.3.1.2 Culture conditions under UV-radiation ........... 13
2.3.1.3 Culture conditions at sub- and supraoptimal temperatures ............ 13
2.3.2 Culture conditions for E. coli ...................................................................... 13
2.4 Plasmids and constructed recombinant vectors ......... 14
2.5 Working with RNA .......................................................................................... 15
2.5.1 Treatment of solutions, glassware and equipment ..................................... 15
2.5.2 Isolation of small and total RNA from T. tenax ........... 15
2.5.2.1 Isopropanol precipitation ............................................................... 15
2.5.2.2 RNeasy Mini Kit (QIAGEN) ........................... 16
TM 2.5.2.3 mirVana miRNA Isolation Kit (Ambion) ...................................... 16
2.5.3 Quantitative and qualitative analysis of RNA ............. 17
2.5.4 Gel electrophoresis of RNA ........................................ 17
2.5.4.1 Denaturing agarose gel electrophoresis ........................................ 17
2.5.4.2 Denaturing polyacrylamide gel electrophoresis ............................. 18
2.5.5 Capillary transfer of RNA to a nylon membrane (Northern blot) ................. 18
2.5.6 Generation of specific, DIG-labelled RNA probes by in vitro transcription . 19
2.5.7 Hybridisation of immobilised total RNA with DIG-labelled probes .............. 21
2.5.7.1 Hybridisation of total RNA with antisense RNA probes ................. 21
2.5.7.2 Hybridisation of small RNA with antisense
oligonucleotide probes .................................................................. 21
2.5.8 Immunological detection of RNA hybrids ................................................... 22
2.5.9 Nuclease assay .......................................................... 22
2.5.10 Electrophoretic Mobility Shift Assay (EMSA) ............ 23 Table of contents II
2.5.11 cDNA synthesis of total RNA from T. tenax ............................................. 23
2.6 Working with DNA .......................................................... 24
2.6.1 Preparation of genomic DNA from T. tenax ............... 24
2.6.2 Preparation of plasmid DNA from E. coli .................................................... 25
2.6.2.1 Preparation of plasmid DNA by alkaline lysis ................................ 25
2.6.2.2 Plasmid preparations with commercially available kits .................. 25
2.6.2.3 Preparation of plasmid-DNA for colony PCR 26
2.6.3 DNA precipitation ....................................................................................... 26
2.6.4 Quantitative and qualitative analysis of DNA ............. 26
2.6.5 Electrophoresis of DNA .............. 26
2.6.5.1 Agarose gel electrophoresis of DNA ............................................. 26
2.6.5.2 Non-denaturing polyacrylamide gel electrophoresis of DNA ......... 27
2.6.6 Purification of DNA fragments .................................... 27
2.6.6.1 Gel extraction of agarose gels....................... 27
2.6.6.2 Purification of PCR fragments ................................ 28
2.6.6.3 Electroelution from polyacrylamide gels ........................................ 28
2.6.7 Polymerase chain reactions (PCR) ............................ 28
2.6.7.1 Amplification of genomic DNA and plasmid DNA .......................... 29
2.6.7.2 Amplification of primary cDNA....................................................... 29
2.6.7.3 PCR mutagenesis ......................................... 30
2.6.7.4 Overlap extension PCR ................................. 30
2.6.8 Enzymatic modification of DNA .. 31
2.6.8.1 Restriction of DNA ......................................... 31
2.6.8.2 5'-dephosphorylation of linearised vector-DNA ............................. 31
2.6.8.3 Ligation ......................................................................................... 31
2.6.9 Transformation ........................... 32
2.6.9.1 Preparation of competent E. coli cells ........................................... 32
2.6.9.2 Transformation of competent E. coli cells ..... 32
2.6.10 Sequencing .............................................................................................. 33
2.6.11 3'-end-labelling of oligonucleotides with digoxigenin ................................ 33
2.6.12 Capillary transfer of DNA to a nylon membrane (Southern blot) .............. 33
2.6.13 RT-PCR Southern blot ............................................................................. 34
2.6.14 Electrophoretic Mobility Shift Assay (EMSA) ............ 34
2.6.15 Immunological detection of DIG-labelled DNA probes ............................. 35
2.6.16 In silico analysis of nucleotide and amino acid sequences ...................... 35
2.7 Biochemical methods .................................................................................... 37
2.7.1 Heterologous expression of T. tenax proteins in E. coli ............................. 37
2.7.2 Preparation, enrichment and purification of the recombinant enzymes ...... 37 Table of contents III
2.7.2.1 Enrichment of the recombinant Csa5 protein ................................ 37
2.7.2.2 Enrichment of the recombinant Csa2 protein 38
2.7.2.3 Enrichment of the recombinant Csa3 protein 38
2.7.2.4 Isolation and solubilisation of proteins from inclusion bodies ........ 40
2.7.2.5 Reconstitution of protein complex CasA1 ..................................... 40
2.7.2.6 Reconstitution of protein complex CasA2 ..... 41
2.7.2.7 Purification of His-tagged recombinant enzymes .......................... 42
2.7.3 Protein quantitation .................................................................................... 42
2.7.4 SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) ............................. 43
2.7.4.1 SDS-PAGE assembling and electrophoresis ................................ 43
2.7.4.2 Coomassie staining ....................................... 43
2.7.4.3 Molecular mass determination of proteins under denaturing
conditions ...................................................................................... 43
2.7.5 Molecular mass determination of proteins under native conditions ............ 44
2.7.6 Determination of the amino acid sequence ................ 44

3 RESULTS ............................................................................................................... 45
3.1 CRISPR loci in T. tenax .................. 45
3.1.1 Identification and features of T. ten