Transcriptional regulation of the central carbohydrate metabolism and synthesis of trehalose in the hyperthermophilic crenarchaeote Thermoproteus tenax [Elektronische Ressource] / vorgelegt von Melanie Zaparty

Sb0034

Sujets

• Biologie

Informations

• Publié par: universitat_duisburg-essen
• Publié le: 01 janvier 2008
• Nombre de lectures: 17
• Langue: Deutsch
• Poids de l'ouvrage: 5 Mo

Extrait

Transcriptional regulation of the central carbohydrate
metabolism and synthesis of trehalose in the hyperthermophilic
crenarchaeote Thermoproteus tenax





Inaugural-Dissertation


zur

Erlangung des Doktorgrades

Dr. rer. nat.

des Fachbereichs

Biologie und Geografie

an der



Universität Duisburg-Essen



vorgelegt von

MELANIE ZAPARTY

aus Essen




April 2007








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

1. Gutachter: Prof. Dr. Reinhard Hensel (Essen)
2. Gutachter: Prof. Dr. Michael Ehrmann (Essen)
3. Gutachter: Prof. Dr. Jörg Soppa (Frankfurt)
Vorsitzende des Prüfungsausschusses: Prof. Dr. Ann Ehrenhofer-Murray (Essen)


Tag der mündlichen Prüfung: 02.08.2007
































































Meinen Eltern -
In Liebe und Dankbarkeit




































Was wir wissen, ist ein Tropfen.
Was wir nicht wissen - ein Ozean.
Isaac Newton

Table of contents


TABLE OF CONTENTS


LIST OF FIGURES...........................................................................................…V
LIST OF TABLES...........................................................................................….. VI
1 INTRODUCTION...........................................................................................…....1
2 MATERIAL AND METHODS ....................................................................…... 14
2.1 Chemicals, enzymes, kits and consumables.............................................….. 14
2.2 Instruments.................................................................................................…. 15
2.3 Strains and growth conditions..................................................................….. 18
2.4 Plasmids and constructed recombinant vectors......................................….. 19
2.5 Biomolecular techniques: Working with DNA........................................…. 22
2.5.1 Preparation of genomic DNA from T. tenax..............................….. 22
2.5.2 Preparation of plasmid DNA from E. coli.................................…. 22
2.5.3 DNA precipitation....................................................................…... 23
2.5.4 Quantitative and qualitative analysis of DNA............................…. 24
2.5.5 Agarose gel electrophoresis of DNA..........................................…. 24
2.5.6 Purification of DNA fragments...................................................…. 25
2.5.7 Polymerase chain reaction (PCR)...............................................…. 25
2.5.7.1 Amplification of genomic DNA and plasmid DNA......…. 26
2.5.7.2 PCR mutagenesis............................................................… 26
2.5.8 Enzymatic modification of DNA.................................................… 26
2.5.8.1 Restriction of DNA..........................................................… 26
2.5.8.2 5’-dephosphorylation of linearised vector-DNA............… 26
2.5.8.3 Ligation...........................................................................… 27
2.5.9 Transformation.............................................................................… 27
2.5.9.1 Preparation of competent E. coli cells................................. 27
2.5.9.2 Transformation of the competent E. coli cells.................… 28
2.5.10 Sequencing...................................................................…............…. 28
2.5.10.1 Automated DNA sequencing........................….............… 28
2.5.10.2 Computer based analysis of nucleotide and amino
acid sequences, and additionally used databases................29
2.5.11 Electrophoretic mobility shift assays (EMSAs).....................…...... 30
2.5.11.1 Generation and 3’-end-labelling of DNA probes with
digoxigenin......................................................…............... 30
2.5.11.2 Incubation assays, electrophoretic separation and
immobilisation of DNA-protein complexes....…............… 31
2.5.12 Immunological detection of DNA-protein complexes…............… 33

2.6 Biomolecular techniques: Working with RNA……………………………..33
2.6.1 Treatment of solutions, glassware and equipment.......................… 33
2.6.2 Isolation of total RNA from T. tenax...........................................… 34
2.6.3 Quantitative and qualitative analysis of RNA..............................… 35
2.6.4 Denaturing agarose gel electrophoresis of RNA..........................… 35
2.6.5 Capillary transfer of RNA to a nylon membrane (Northern blot)… 36

I Table of contents


2.6.6 Hybridisation of immobilised total RNA with radioactively
labelled specific RNA probes.....................................................…. 37
322.6.6.1 Generation of specific, [α P]-labelled antisense RNA
probes by in vitro transcription............................................ 37
322.6.6.2 Hybridisation of RNA with [α- P]-CTP labelled
probes................................................................................... 39
2.6.6.3 Detection of RNA-RNA hybrids (Autoradiography).......... 39

2.7 Design, fabrication and application of the cDNA microarray.................… 40

2.7.1 Microarray probe generation using PCR..........................................25
2.7.2 Printing of the microarrays...............................................................26
2.7.3 Post-processing of the slides.............................................................42
2.7.4 Preparation of the internal standard (rpoS) by in vitro
transcription.................................................................................….42
2.7.5 Target generation: Labelling and cDNA synthesis of total RNA
from T. tenax.................................................................................... 43
2.7.6 Hybridisation of the labelled cDNA to the microarray................… 44
2.7.7 Scanning of the microarrays, data processing and analysis............. 44

2. 8 Biochemical methods...................................................................................... 46

2.8.1 Heterologous expression of the T. tenax TPSP, GT, MsC,
LrP1 and HP5 in E. coli........................................................……... 46
2.8.2 Expression of the T. tenax MsC in Sulfolobus solfataricus.....…… 47
2.8.3 Preparation, enrichment and purification of the recombinant
enzymes............................................................................................ 47
2.8.3.1 Enrichment of the recombinant TPSP.................................. 47
2.8.3.2 In vitro reconstitution of the GT from inclusion bodies..… 48
2.8.3.3 Isolation of the recombinant MsC from S. solfataricus…... 49
2.8.3.4 Enrichment of the recombinant Lrp1 and HP5
for EMSAs................................................................................…... 49
2.8.3.5 Purification of His-tagged recombinant enzymes………… 49
2.8.4 Determination of the enzyme activities of the recombinant TPSP.. 50
2.8.5 Measurements in crude extracts of T. tenax...............................….. 51
2.8.6 Thin layer chromatography (TLC) ..............................................… 51
2.8.7 Analytical protein methods...........................................................…52
2.8.7.1 Protein quantitation...........................................................…52
2.8.7.2 SDS Polyacrylamide gel electrophoresis (PAGE) ..........… 52
2.8.7.3 Molecular mass determination under denaturing
conditions..........................................................…..........…..53
2.8.7.4 Electrotransfer of separated protein species to a
membrane (Western blot) ..................................…..........… 54
2.8.7.5 Determination of the N-terminal amino acid sequence.…...54


3 RESULTS.........................................................................................................….. 55
3.1 Transcriptional profiling of CCM genes using cDNA microarrays..…….. 55
3.1.1 Microarray fabrication.............................................……………… 55

II Table of contents


3.1.1.1 Probe generation………………………………………….. 55
3.1.1.2 Printing a