High-throughput metabolome analysis of transposon mutants of Corynebacterium glutamicum and quenching of microbial metabolism [Elektronische Ressource] / von Jana Spura

Jbo

Sujets

• Biologie

Informations

• Publié par: technische_universitat_carolo-wilhelmina_zu_braunschweig
• Publié le: 01 janvier 2009
• Nombre de lectures: 38
• Poids de l'ouvrage: 4 Mo

Extrait

TECHNISCHE UNIVERSITÄT
CAROLO-WILHELMINA
ZU BRAUNSCHWEIG
High-throughput metabolome analysis of transposon
mutants of Corynebacterium glutamicum and
quenching of microbial metabolism
Von der Fakultät für Lebenswissenschaften
der Technischen Universität Carolo-Wilhelmina
zu Braunschweig
zur Erlangung des Grades einer
Doktorin der Naturwissenschaften
(Dr. rer. nat.)
genehmigte
D i s s e r t a t i o n
von Jana Spura
aus Torgau1. Referentin oder Referent: Professor Dr. Dietmar Schomburg
2. Referentin oder Referent: Professor Dr. Dieter Jahn
eingereicht am: 15.12.2008
mündliche Prüfung (Disputation) am: 24.02.2009
Druckjahr 2009Vorveröffentlichungen der Dissertation
Teilergebnisse aus dieser Arbeit wurden mit Genehmigung der Fakultät für
Lebenswissenschaften, vertreten durch den Mentor der Arbeit, in folgenden
Beiträgen vorab veröffentlicht:
Publikationen
Börner, J., Buchinger, S. & Schomburg, D. A high-throughput method for microbial
metabolome analysis using gas chromatography/mass spectrometry. Analytical
Biochemistry 367: 143-151 (2007)
Spura, J. & Schomburg D. Hochdurchsatzmetabolomanalyse von Mikro-
organismen. Laborwelt 6: 21-24 (2008)
Tagungsbeiträge
Börner, J., Buchinger, S. & Schomburg, D. High-throughput method for microbial
metabolome analysis. (Poster) European BioPerspectives, Köln (2007).
Börner, J., Buchinger, S. & Schomburg, D. High-throughput method for microbial
metabolome analysis. (Poster) ProkaGENOMICS, Göttingen (2007).
Spura, J., Reimer, L., Lühr, T. & Schomburg, D. Screening of transposon mutants
of Corynebacterium glutamicum using a high-throughput method for microbial
metabolome analysis. (Vortrag) European BioPerspectives, Hannover (2008).
Reimer, L., Spura, J., Wieloch, P., Schreiber, K. & Schomburg, D Quenching of
different microorganisms for metabolome analysis using an ethanol/sodium
chloride-quenchingsolution (Poster) European BioPerspectives, Hannover (2008). Table of contents I
Table of contents
Abstract.................................................................................................................................III
Kurzzusammenfassung.........................................................................................................IV
Abbreviations & Formula......................................................................................................V
1 Introduction.....................................................................................................................1
1.1 Systems biology.........................................................................................................1
1.1.1 Genomics............................................................................................................1
1.1.2 Transcriptomics..................................................................................................2
1.1.3 Proteomics..........................................................................................................2
1.1.4 Metabolomics.....................................................................................................3
1.2 Metabolomics in functional genomics.......................................................................3
1.2.1 Methods in metabolome analysis.......................................................................4
1.2.2 Pre-analytical sample preparation for metabolome analysis with
Gas Chromatography/Mass Spectrometry.......................................................15
1.2.3 High-throughput performances in metabolome analysis.................................19
1.2.4 Transposon mutagenesis and the construction of a mutant bank.....................19
1.3 Model organisms......................................................................................................21
1.3.1 Corynebacterium glutamicum..........................................................................21
1.3.2 Escherichia coli................................................................................................23
1.3.3 Saccharomyces cerevisiae................................................................................24
1.4 Thesis aims...............................................................................................................26
2 Material & Methods......................................................................................................27
2.1 Used chemicals and machines.................................................................................27
2.2 Software and libraries..............................................................................................31
2.3 Used bacteria strains................................................................................................33
2.4 Applied kits..............................................................................................................33
2.5 Statistic and calculation of errors.............................................................................34
2.6 Media, buffer and solutions.....................................................................................35
2.7 Standard metabolome analysis ................................................................................46
2.7.1 Cultivation in shaking flasks............................................................................46
2.7.2 Pre-analytical sample preparation....................................................................47II Table of contents
2.7.3 GC/MS analysis and data processing...............................................................49
2.8 High-throughput metabolome analysis....................................................................50
2.8.1 Cultivation in micro titer plates........................................................................50
2.8.2 Semi-automatic sample preparation.................................................................51
2.8.3 GC/MS analysis and data processing...............................................................53
2.9 Quenching of microorganisms.........53
2.9.1 Ethanol quenching (EQ)...................................................................................55
2.9.2 Methanol quenching (MQ)...............................................................................56
2.9.3 Cold glycerol saline quenching (GSQ)............................................................56
2.10 Plasmid rescue technique.......57
3 Results & Discussion.....................................................................................................62
3.1 Development of a high-throughput method for metabolome analysis....................62
3.1.1 Method development........................................................................................62
3.1.2 Method evaluation............................................................................................72
3.2 Screening of transposon mutants.............................................................................75
3.2.1 Growth of analyzed transposon mutants..........................................................75
3.2.2 Development of suitable screening criteria......................................................77
3.2.3 Hierarchical clustering of the growth-deficient mutants..................................92
3.2.4 Investigation of changes in metabolite levels..................................................94
3.2.5 Identification of the transposon insertion site of some mutants.......................97
3.3 Comparison of different quenching methods.........................................................101
3.3.1 Refinement of the ethanol quenching method...............................................101
3.3.2 Difficulties in the cold glycerol saline quenching performance....................106
3.3.3 Quenching of Escherichia coli.......................................................................107
3.3.4 Quenching of Corynebacterium glutamicum.................................................116
3.3.5 Quenching of Saccharomyces cerevisiae.......................................................123
4 Summary......................................................................................................................130
5 Outlook.........................................................................................................................132
6 References.....................................................................................................................133
7 Appendix.......................................................................................................................142 Abstract III
Abstract
A suitable method is the prerequisite for comprehensive screening performances.
Therefore, an analytical method for high-throughput metabolome analysis on basis of our
well established standard method was developed. Fast metabolome analysis was done
using gas chromatography/mass spectrometry. Two major advantages were parallelization
of operations and partial automation. In short this means a clear reduction of time needed
for the pre-analytical steps. The standard error between independent samples of the model
organism Corynebact