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Table of contents
Introduction and context of the study
Part 1: Literature review
1 Population heterogeneity
1.1 Form of heterogeneity
1.2 Cause of heterogeneity
1.2.1 Gene expression stochasticity
1.2.2 Cell cycle
1.2.3 Age distribution
1.2.4 Bet-hedging
1.2.5 Extracellular micro-environment
1.3 Heterogeneity analysis at single-cell level
1.3.1 Single-cell isolation / individualization
1.3.2 Single-cell analysis
2 Plasmid stability
2.1 Causes and consequences of plasmid loss
2.1.1 Metabolic load of recombinant plasmids
2.1.2 Segregational plasmid stability
2.1.3 Structural plasmid stability
2.1.4 Mating
2.1.5 Importance of the plasmid-host interaction
2.2 Segregational plasmid stability monitoring
2.2.1 Plate count
2.2.2 Reporter protein expression
2.2.3 Plasmid copy number determination
2.3 Plasmid stabilization strategies
2.3.1 Recombinant plasmid genetic construction
2.3.2 Mechanisms of plasmid stable maintenance
3 Study model: isopropanol production by Cupriavidus necator
3.1 Genome description
3.1.1 The two chromosomes
3.1.2 The megaplasmid
3.2 Metabolism description
3.2.1 Heterotrophic metabolism
3.2.2 Autotrophic metabolism
3.2.3 Central carbon metabolism
3.2.4 Lithotrophic metabolism
3.2.5 Respiratory mechanism
3.2.6 PHB biosynthesis
3.3 C. necator as a recombinant bioproduction platform
3.4 Population heterogeneity in Cupriavidus necator
3.5 Plasmid stability strategies in Cupriavidus necator
3.6 Isopropanol production by recombinant Cupriavidus necator strains
3.6.1 Natural biological production
3.6.2 Recombinant biological production
4 Conclusion and objective of the study
Part 2: Material and methods
1 Strains, plasmids and media
1.1 Strains
1.2 Media
1.2.1 Rich media
1.2.2 Mineral media
1.3 Plasmids
1.3.1 Description of plasmids
1.3.2 Plasmid construction
2 Culture conditions
2.1 Glycerol stock preparation
2.2 Preculture scheme and flask cultivation on fructose
2.2.1 Preculture scheme
2.2.2 Flask cultivations on fructose
2.2.3 Plasmid curing subcultures in flasks
2.2.4 Bioreactor inoculation
2.3 Bioreactor fermentations
2.3.1 Experimental set-up
2.3.2 Process control and regulation
2.3.3 Gas analysis
2.3.4 Batch cultivations
2.3.5 Fed-Batch cultivations
2.3.6 Continuous cultures
2.3.7 Sampling procedure description
3 Analytical procedure
3.1 Biomass characterization
3.1.1 Optical density
3.1.2 Cell Dry Weight
3.1.3 Optical microscopy
3.2 Plate count
3.3 Flow Cytometry
3.3.1 Working principle of the flow cytometer
3.3.2 Experimental protocol for population heterogeneity assessment
3.4 Fluorescence Activated Cell Sorting (FACS)
3.5 Fluorescence measurement in the medium
3.6 Determination of fructose, organic acids and ammonium concentrations
3.6.1 High-performance liquid chromatography (HPLC)
3.6.2 Gas chromatography (GC)
3.6.3 High Pressure Ionic Chromatography (HPIC)
4 Methodologies for data treatment
4.1 Rate expression for gas-phase reactions
4.1.1 Nitrogen balance
4.1.2 Dioxygen balance
4.1.3 Carbon dioxide balance
4.2 Volume determination during fed-batch cultures
4.3 Rate expressions for liquid-phase reactions
4.4 Determination of instantaneous and overall yields
4.5 Carbon and elemental balances
4.5.1 Carbon balance
4.5.2 Nitrogen balance
4.5.3 Elemental balance
4.6 Smoothing of experimental data
4.7 Statistical analysis: Normality of distribution functions by BoxPlot representation
Part 3: Results and discussion
Chapter 1: Plasmid expression stability during heterologous isopropanol production in fed-batch bioreactor1.1 Abstract
1.2 Introduction
1.3 Material and method
1.4 Results
1.5 Discussion
1.6 Results synthesis
Chapter 2: Identification of heterologous subpopulations from a pure culture in a bioreactor
Subchapter 1: Plasmid expression level heterogeneity monitoring via heterologeous eGFP production at the single-cell level in Cupriavidus necator
Subchapter 2: Study of plasmid expression level heterogeneity under plasmid-curing like conditions
2.2.1 Abstract
2.2.2 Introduction
2.2.3 Material and Methods
2.2.4 Results
2.2.5 Discussion
2.2.6 Results synthesis
Subchapter 3: Investigation of the robustness of Cupriavidus necator engineered strains during fed-batch cultures
2.3.1 Abstract
2.3.2 Introduction
2.3.3 Material and methods
2.3.4 Results
2.3.5 Discussion
2.3.6 Results synthesis
Chapter 3: Plasmid expression level heterogeneity during heterogeneous isopropanol production studied by an eGFP monitoring system in fed-batch bioreactor
3.1 Abstract
3.2 Introduction
3.3 Material and method
3.4 Results
3.5 Discussion
3.6 Results synthesis
Part 4: General discussion, conclusions and perspectives
References
