Characterization of SsoSSB, Sso1450, Sso2001 proteins and analysis of CRISPR and cas genes from Sulfolobus solfataricus P2 [Elektronische Ressource] / vorgelegt von Dong Han

Sujets

• Biologie

Informations

• Publié par: universitat_bayreuth
• Publié le: 01 janvier 2008
• Nombre de lectures: 42
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

Characterization of SsoSSB, Sso1450,
Sso2001 Proteins and Analysis of CRISPR
and cas Genes from Sulfolobus solfataricus P2

Dissertation

zur Erlangung des Grades Doktor der Naturwissenschaften
- Dr. rer. nat.-

der Fakultät für Biologie, Chemie und Geowissenschaften
der Universität Bayreuth



vorlegt von
Dong Han
aus Shandong, China


Bayreuth 2007 Die vorliegende Arbeit wurde in der Zeit von Juni 2001 bis März 2007 am Lehrstuhl für
Biochemie der Universität Bayreuth unter der Leitung von Herrn Prof Dr. Gerhard
Krauss angefertigt.

Vollständiger Abdruck der von der Fakultät Biologie, Chemie und Geowissenschaften
der Universität Bayreuth genehmigten Dissertation zu Erlangung des Grades eines
Doktors der Naturwissenschaften (Dr. rer. nat.).














Promotionsgesuch eingereicht am : January16, 2008
Tag des wissenschaftlichen Kolloquiums: May 07, 2008


Prüfungsausschuss:

Prof. Dr. Gerhard Krauss (Erster Gutachter)
Prof. Dr. Wolfgang Schumann (Zweiter Gutachter)
Prof. Dr. Carlo Unverzagt (Vorsitzender)
Prof. Dr. Franz Meußdoerfer


0HTable of contents
Table of contents

TABLE OF CONTENTS ..............................................................................................................................I
ABBREVIATIONS.......................................................................................................................................V
1. INTRODUCTION ................................................................................................................1
1.1 ARCHAEA SULFOLOBUS SOLFATARICUS P2 STRAIN..................................................................................1
1.1.1 Archaea: ubiquitous but unique....................................................................................................1
1.1.2 Sulfolobus solfataricus, a model system in crenarchaea...............................................................3
1.2 SSBS......................................................................................................................................................4
1.2.1 General introduction of SSBs........................................................................................................4
1.2.2 Bacterial and human mitochondrial SSBs ....................................................................................5
1.2.3 Replication protein A (RPA), the eukaryotic SSBs........................................................................6
1.2.4 Archaeal SSB, the ancient SSB?.........8
1.2.5 Other SSBs ..................................................................................................................................10
1.3 CRISPR AND CRISPR-ASSOCIATED PROTEINS11
1.3.1 General introduction of CRISPR.....11
1.3.2 CRISPR-associated proteins .......................................................................................................12
1.3.3 The biological roles of CRISPRs and Cas proteins ....................................................................13
1.3.4 CASS in Sulfolobus solfataricus..................................................................................................15
1.3.5 Prospect ......................................................................................................................................17
1.4 AIM OF THE PRESENT WORK18
2. MATERIALS AND METHODS............................................................................................................20
2.1 MATERIALS .........................................................................................................................................20
2.1.1 Chemicals, enzymes and proteins.....20
2.1.1.1 Chemicals............................................................................................................................................ 20
2.1.1.2 Enzymes and proteins.......................................................................................................................... 20
2.1.2 Bacterial and archaeal strains, media and antibiotics ...............................................................21
2.1.2.1 Bacterial strains................................................................................................................................... 21
2.1.2.2 Media, inducer and antibiotics ............................................................................................................ 21
2.1.3 Plasmids and phage.............21
2.1.4 Oligonucleotides and tRNAs .......................................................................................................22
2.1.4.1 PCR primers........................................................................................................................................ 22
2.1.4.2 Primers for mutation............................................................................................................................ 23
2.1.4.3 Substrates for SsoSSB ......................................................................................................................... 23
2.1.4.4 Substrates for nuclease assay............................................................................................................... 24
2.1.4.5 Substrates for Sso1450C6H................................................................................................................. 24
2.1.5 Buffers and solutions...................................................................................................................25
2.1.6 Commercial kits ..........................................................................................................................27
2.1.7 Instruments and materials...........................................................................................................27
2.1.8 Chromatographic materials........28
2.1.9 Softwares.....................................................................................................................................28
2.2 STANDARD METHODS ..........................................................................................................................28
2.2.1 Spectrophotometric determination..............................................................................................28
2.2.1.1 Determination of protein concentration............................................................................................... 28
2.2.1.2 Determination of nucleic acid concentration....................................................................................... 29
2.2.1.3 Determination of bacterial cell density................................................................................................ 29
2.2.2 Gel electrophoresis .....................................................................................................................29
2.2.2.1 Agarose gel electrophoresis................................................................................................................. 29
2.2.2.2 Native polyacrylamide gel electrophoresis.......................................................................................... 30
2.2.2.3 Denaturing polyacrylamide gel electrophoresis .................................................................................. 30
2.2.2.4 SDS polyacrylamide gel electrophoresis (SDS PAGE)....................................................................... 30
2.2.3 Detection of radioactively labeled nucleic acids in gel...............................................................31
I
21H15H4H11H30H16H12H25H13H17H14H29H26H5H3H46H42H49H23H6H19H7H47H8H45H9H28H44H27H39H2H10H50H31H43H32H41H33H24H48H22H38H20H34H18H35H51H36H37H40HTable of contents

2.2.4 Detection of unlabeled nucleic acids in gel.................................................................................31
2.2.5 Staining of protein gels ...............................................................................................................31
2.3 MOLECULAR BIOLOGY METHODS.........................................................................................................31
2.3.1 Preparation and transformation of competent cells....................................................................31
2.3.2 Culture of bacterial strains .........................................................................................................32
2.3.3 Extraction of nucleic acids with phenol:chloroform...................................................................33
2.3.4 Nucleic acid precipitation by ethanol .........................................................................................33
2.3.5 Dephosphorylation of nucleic acid .............................................................................................33
322.3.6 5’-end labeling of oligonucleotides by [ γ- p]-ATP33
2.3.7 Hybridization of oligonucleotides.......34
2.3.8 Small scale