Engineering Tuneable Gene Circuits in Yeast

Inhaltsverzeichnis (Table of Contents)

• Introduction
• Aims and Objectives
• The Yeast Saccharomyces cerevisiae
• Yeast Mating
• Pheromone Receptor-G-protein Coupling
• Pheromone-Induced G-protein Activation
• The MAP Kinase Cascade
• Ste11, Ste7 and Fus3
• Ste 12 and The Pheromone Response Element
• Switching Off The Pheromone Response
• Modelling The Mating Pathway
• Chen et al (2000): Kinetic Analysis of Budding Yeast Cell Cycle Model
• Yi et al G-Protein Model
• Hao et al RGS Protein Pheromone Desensitization Model
• The Kofahl and Klipp Yeast Pheromone Pathway Model
• Modelling tools
• Copasi
• XPPAUT
• Cytoscape
• Mathematical Programming Languages
• Scripting Languages
• SBML
• Metabolic Control Analysis
• Parameter Estimation
• Signal to Noise Ratio
• Synthetic Biology
• Synthetic Oscillators
• Transcription Cascades
• Synthetic Switches
• Riboswitches
• Application of Synthetic Biology
• Project Overview
• Materials and Methods
• Plasmids
• Primers
• Yeast & Bacterial Strains
• Yeast Growth Conditions
• Bacterial Growth Conditions
• Transformation of competent E. coli TOP10 cells
• MINIPrep Plasmid Purification
• Manual Miniprep Plasmid Purification Protocol
• Reagents
• 25% sucrose
• Lysozyme
• Triton Lytic Mix
• Plasmid DNA Restriction digest
• Analytical Plasmid DNA Digest
• Preparative Digest
• Cranenburgh Ligation Method
• Primer Design
• PCR
• Colony PCR Protocol
• Genomic DNA Extraction
• Extraction Buffer
• Site Directed Mutagenesis Protocol
• Site Directed Mutagenesis PCR Reaction Program
• Phosphorylation and Annealing of Synthetic Oligonucleotides
• Agarose Gel Electrophoresis
• TAE buffer - 5 Litre, 10x stock
• Preparation of DNA loading dye
• Yeast Transformation
• Preparation of Solutions and Growth Media for Yeast Transformation
• Preparation of 10x LiAc and 10X TE solution for yeast transformation
• Preparation of 20ml PEG/LiAc/TE solution
• Preparation of YP agar
• Yeast transformation protocol
• Yeast Protein Extraction
• Lysis buffer
• SDS Sample buffer
• Preparation of SDS PAGE Protein Gels
• Western blotting
• Polyacrylamide gel electrophoresis protocol
• Western Blot Transfer protocol
• Antibody binding
• Western Blot Imaging
• Alkaline Phosphatase Protocol
• Quantification of Western Blot Images
• DNA Sequence Alignment
• DNA Primer Design
• Pheromone Induction of Yeast Cells for Luminescence Assay
• Optical Density Measurements
• Cellometer Cell Measurements
• Yeast Growth Rate Measurements
• Yeast in situ Luciferase Assay
• Real-time Quantitative PCR (RT-qPCR)
• RT-qPCR Primer Design
• mRNA extraction and purification
• Turbo DNase protocol
• Reverse Transcriptase protocol
• RT-qPCR protocol
• Mathematical Modelling
• Metabolic Control Analysis
• Sensitivity Analysis
• Metabolic Control Analysis
• Signal to Noise Ratio
• Parameter Estimation
• Dissertation
• Results - Circuit Construction
• Introduction
• The Iron Responsive Element-Binding Protein
• The LexA DNA Binding Protein
• Yeast Promoters
• Circuit Overview
• Design overview
• Component Interactions
• Overview of Luciferase Gene Expression Tuning
• Construction of the Reporter Plasmid
• The Luciferase Reporter Gene
• Insertion of the Iron Response Element
• Construction of the Repressor Plasmid
• Cloning the Iron Response Protein Gene
• TRP1 promoter strategy
• TEF1 promoter strategy
• DCD1 promoter strategy
• Insertion of LexA Operator Control Sequences
• Cloning the IRP PEST Degradation Tag
• Construction of the De-Repressor Plasmid
• Conclusion
• Results - Circuit Characterization
• Introduction
• Growth Rate Investigation
• Luminescence Measurement
• Luciferase Signal to Noise Ratio
• Protein Quantification
• mRNA Quantification
• qPCR Housekeeping Gene Selection
• Primer Validation
• Sample Preparation
• pDCD1 Circuit qPCR Analysis
• pTEF1 Circuit qPCR Analysis
• pDCD1-PEST Circuit qPCR Analysis
• pTEF1 Circuit qPCR Analysis
• qPCR Analysis Summary
• Conclusion
• Modelling
• Introduction
• Modelling Eukaryotic Signal Cascades
• A Revised Mating Pathway Model
• Simulation Results

Zielsetzung und Themenschwerpunkte (Objectives and Key Themes)

This thesis investigates the design and construction of tuneable gene circuits in yeast, aiming to develop a robust and reliable platform for synthetic biology applications. The work focuses on manipulating the yeast mating pathway, a well-characterized and highly regulated cellular process, to create synthetic gene circuits with predictable and controllable outputs.

• Engineering of gene circuits in yeast
• Control and tuning of gene expression
• Application of synthetic biology principles
• Mathematical modeling and analysis of gene circuits
• Potential applications in biotechnology and medicine

Zusammenfassung der Kapitel (Chapter Summaries)

• Introduction: This chapter provides a comprehensive overview of the yeast mating pathway and its relevance to the development of synthetic gene circuits. It outlines the aims and objectives of the research, highlighting the need for robust and controllable gene circuits in synthetic biology. It also reviews existing models of the mating pathway and discusses the importance of mathematical modeling in understanding and predicting circuit behavior.
• Materials and Methods: This chapter details the experimental methods used throughout the research, including techniques for cloning, gene manipulation, yeast transformation, protein and mRNA quantification, and mathematical modeling. It provides a detailed description of the protocols and reagents used to construct and characterize the gene circuits.
• Results - Circuit Construction: This chapter describes the design and construction of the tuneable gene circuits, focusing on the selection and integration of key components, including the iron responsive element-binding protein, the LexA DNA binding protein, and yeast promoters. It discusses the strategies employed to control gene expression and ensure circuit functionality.
• Results - Circuit Characterization: This chapter presents the experimental results obtained from characterizing the constructed gene circuits. It investigates the effects of various circuit parameters on gene expression, including growth rate, luminescence output, protein levels, and mRNA levels. It also analyzes the data using mathematical modeling techniques to gain a deeper understanding of circuit behavior.
• Modelling: This chapter focuses on the mathematical modeling of the yeast mating pathway and the engineered gene circuits. It presents a revised model of the mating pathway that incorporates the key elements of the constructed circuits. The model is used to simulate circuit behavior and explore the impact of different parameters on gene expression.

Schlüsselwörter (Keywords)

This thesis delves into the realm of synthetic biology, specifically focusing on the development of tuneable gene circuits in the yeast Saccharomyces cerevisiae. Key themes include the engineering of gene expression, the manipulation of the yeast mating pathway, and the application of mathematical modeling to understand and predict circuit behavior. The work also explores the potential applications of these gene circuits in various fields, including biotechnology and medicine.