Engineering Tuneable Gene Circuits in Yeast

Table of Contents

1 Introduction

1.1 Aims and Objectives

1.2 The Yeast Saccharomyces cerevisiae

1.2.1 Yeast Mating

1.2.2 Pheromone Receptor-G-protein Coupling

1.2.3 Pheromone-Induced G-protein Activation

1.2.4 The MAP Kinase Cascade

1.2.4.1 Ste11, Ste7 and Fus3

1.2.4.2 Ste12 and The Pheromone Response Element

1.3 Switching Off The Pheromone Response

1.4 Modelling The Mating Pathway

1.4.1 Chen et al (2000): Kinetic Analysis of Budding Yeast Cell Cycle Model

1.4.2 Yi et al G-Protein Model

1.4.3 Hao et al RGS Protein Pheromone Desensitization Model

1.4.4 The Kofahl and Klipp Yeast Pheromone Pathway Model.

1.4.5 Modelling tools

1.4.5.1 Copasi

1.4.5.2 XPPAUT

1.4.5.3 Cytoscape

1.4.5.4 Mathematical Programming Languages

1.4.5.5 Scripting Languages

1.4.5.6 SBML

1.4.6 Metabolic Control Analysis

1.4.7 Parameter Estimation

1.4.8 Signal to Noise Ratio

1.5 Synthetic Biology

1.5.1 Transcription Cascades

1.5.2 Synthetic Oscillators

1.5.3 Synthetic Switches

1.5.4 Riboswitches

1.5.5 Application of Synthetic Biology

1.5.6 Project Overview

2 Materials and Methods

2.1 Plasmids

2.2 Primers

2.3 Yeast & Bacterial Strains

2.4 Yeast Growth Conditions

2.5 Bacterial Growth Conditions

2.6 Transformation of competent E. coli TOP10 cells

2.7 MINIPrep Plasmid Purification

2.8 Manual Miniprep Plasmid Purification Protocol

2.8.1 Reagents

2.8.1.1 25% sucrose

2.8.1.2 Lysozyme

2.8.1.3 Triton Lytic Mix

2.9 Plasmid DNA Restriction digest

2.9.1 Analytical Plasmid DNA Digest

2.9.2 Preparative Digest

2.10 Cranenburgh Ligation Method

2.11 Primer Design

2.12 PCR

2.13 Colony PCR Protocol

2.14 Genomic DNA Extraction

2.14.1 Extraction Buffer

2.15 Site Directed Mutagenesis Protocol

2.15.1 Site Directed Mutagenesis PCR Reaction Program

2.16 Phosphorylation and Annealing of Synthetic Oligonucleotides

2.17 Agarose Gel Electrophoresis

2.17.1 TAE buffer - 5 Litre, 10x stock

2.17.2 Preparation of DNA loading dye

2.18 Yeast Transformation

2.18.1 Preparation of Solutions and Growth Media for Yeast Transformation

2.18.1.1 Preparation of 10x LiAc and 10X TE solution for yeast transformation

2.18.1.2 Preparation of 20ml PEG/LiAc/TE solution

2.18.1.3 Preparation of YP agar

2.18.2 Yeast transformation protocol

2.19 Yeast Protein Extraction

2.19.1 Lysis buffer

2.19.2 SDS Sample buffer

2.19.3 Preparation of SDS PAGE Protein Gels

2.20 Western blotting

2.20.1 Polyacrylamide gel electrophoresis protocol

2.20.2 Western Blot Transfer protocol

2.20.3 Antibody binding

2.20.4 Western Blot Imaging

2.20.5 Alkaline Phosphatase Protocol

2.20.6 Quantification of Western Blot Images

2.21 DNA Sequence Alignment

2.22 DNA Primer Design

2.23 Pheromone Induction of Yeast Cells for Luminescence Assay

2.24 Optical Density Measurements

2.25 Cellometer Cell Measurements

2.26 Yeast Growth Rate Measurements

2.27 Yeast in situ Luciferase Assay

2.28 Real-time Quantitative PCR (RT-qPCR)

2.28.1 RT-qPCR Primer Design

2.28.2 mRNA extraction and purification

2.28.3 Turbo DNase protocol

2.28.4 Reverse Transcriptase protocol

2.28.5 RT-qPCR protocol

2.29 Mathematical Modelling

2.29.1 Metabolic Control Analysis

2.29.2 Sensitivity Analysis

2.29.3 Metabolic Control Analysis

2.29.4 Signal to Noise Ratio

2.29.5 Parameter Estimation

2.30 Dissertation

3 Results - Circuit Construction

3.1 Introduction

3.1.1 The Iron Responsive Element-Binding Protein

3.1.2 The LexA DNA Binding Protein

3.1.3 Yeast Promoters

3.2 Circuit Overview

3.2.1 Design overview

3.2.2 Component Interactions

3.2.3 Overview of Luciferase Gene Expression Tuning

3.3 Construction of the Reporter Plasmid

3.3.1 The Luciferase Reporter Gene

3.4 Insertion of the Iron Response Element

3.5 Construction of the Repressor Plasmid

3.5.1 Cloning the Iron Response Protein Gene

3.5.1.1 TRP1 promoter strategy

3.5.1.2 DCD1 promoter strategy

3.5.1.3 TEF1 promoter strategy

3.5.2 Insertion of LexA Operator Control Sequences

3.5.3 Cloning the IRP PEST Degradation Tag

3.6 Construction of the De-Repressor Plasmid

3.7 Conclusion

4 Results - Circuit Characterization

4.1 Introduction

4.2 Growth Rate Investigation

4.3 Luminescence Measurement

4.3.1 Luciferase Signal to Noise Ratio

4.4 Protein Quantification

4.5 mRNA Quantification

4.5.1 qPCR Housekeeping Gene Selection

4.5.2 Primer Validation

4.5.3 Sample Preparation

4.5.4 pDCD1 Circuit qPCR Analysis

4.5.5 pTEF1 Circuit qPCR Analysis

4.5.6 pDCD1-PEST Circuit qPCR Analysis

4.5.7 pTEF1 Circuit qPCR Analysis

4.5.8 qPCR Analysis Summary

4.6 Conclusion

5 Modelling

5.1 Introduction

5.2 Modelling Eukaryotic Signal Cascades

5.2.1 A Revised Mating Pathway Model

5.2.1.1 Simulation Results

5.3 Modelling the Gene Circuit

5.4 Model Parameterisation

5.4.1 Further Parameterisation and the Final Model

5.5 Stochastic Simulation of the Gene Circuit

5.6 Parameter Estimation

6 Discussion

6.1 Introduction

6.2 Design and Development

6.3 Characterisation

6.4 Noise

6.5 Modelling

6.6 Summary and Further Work

6.7 Conclusion

A Appendix

A.1 Sequences

A.1.1 Iron Response Element (IRE) Nucleotide Sequence

A.1.2 PFUS1-IRE-Luciferase Nucleotide Sequence

A.1.3 PFUS1-LexA Nucleotide Sequence

A.1.4 Cln2 Protein Sequence

A.1.5 PEST region nucleotide sequence

A.1.6 Iron Response Protein (IRP) Nucleotide Sequence

A.1.7 IRPPEST Nucleotide Sequence

A.1.8 IRPPEST protein sequence

A.1.9 LexA Operator, DCD1 promoter, and IRP Nucleotide Sequence

A.1.10 LexA Operator, TEF1 promoter, and IRP Nucleotide Sequence

B Appendix

B.1 Python script for processing Copasi stochastic data

Research Objectives and Key Topics

This project aims to engineer a novel, tuneable gene circuit in the budding yeast Saccharomyces cerevisiae to study the dynamics of transcriptional and translational repression. The research investigates whether combining these two modes of repression can effectively suppress basal expression of a reporter gene while allowing for a robust, pheromone-induced activation, thereby increasing the signal-to-noise ratio in synthetic gene circuitry.

• Construction and characterization of a synthetic gene circuit using inducible FUS1 promoters in yeast.
• Dual-level repression strategy utilizing the human iron response protein (IRP) and the bacterial LexA repressor.
• Integration of in silico mathematical modelling with in vivo experiments to predict and optimize circuit performance.
• Investigation of metabolic burden and noise reduction through promoter tuning and protein degradation tags (PEST sequences).
• Development of quantitative tools, including RT-qPCR and luminescence assays, to measure dynamic circuit output.

Excerpt from the Book

Summary of Chapters

1 Introduction: Provides an overview of the yeast pheromone response pathway and principles of synthetic biology, establishing the foundation for designing gene circuits that utilize these natural biological components.

2 Materials and Methods: Details the laboratory procedures used for plasmid construction, yeast transformation, protein and mRNA quantification, and the mathematical modelling approaches employed.

3 Results - Circuit Construction: Describes the design and physical assembly of the three novel yeast expression plasmids that constitute the synthetic circuit.

4 Results - Circuit Characterization: Presents the experimental data on growth rates, luminescence output, and protein/mRNA expression levels, evaluating how the circuit performs in induced versus non-induced conditions.

5 Modelling: Outlines the computational modelling framework, including parameter estimation and stochastic simulations, used to refine and predict the circuit’s complex dynamic behaviour.

6 Discussion: Analyzes the experimental and modelling findings, evaluating the success of the circuit design and identifying areas for further optimization and future development.

Keywords

Synthetic Biology, Saccharomyces cerevisiae, Gene Circuits, Pheromone Response Pathway, Luciferase, Translational Repression, Transcriptional Repression, Mathematical Modelling, Metabolic Control Analysis, Signal-to-Noise Ratio, Promoter Tuning, PEST Tag, LexA, IRP, System Dynamics

Frequently Asked Questions

What is the core focus of this research?

The research focuses on the design and construction of a novel synthetic gene circuit in yeast that integrates transcriptional and translational repression to create a more efficient "switch" for gene expression.

What are the primary thematic areas?

The work combines synthetic biology, systems biology, computational modelling, molecular cloning, and protein/mRNA quantification techniques in the model organism Saccharomyces cerevisiae.

What is the main research objective?

The main objective is to build a gene circuit that reduces basal (background) noise and increases the fold-change response to an external environmental stimulus (pheromone) compared to existing systems.

What scientific methods were employed?

The project utilized a dual-approach method: wet-lab molecular biology (plasmid construction, Western blotting, RT-qPCR, luminescence assays) and in silico computational modelling (Metabolic Control Analysis, stochastic simulation, parameter estimation).

What topics are covered in the main body?

The main body covers the theoretical basis of the mating pathway, the construction of reporter/repressor/de-repressor plasmids, the experimental characterization of these circuits, and the iterative loop between mathematical prediction and physical experimentation.

Which keywords define this work?

Key terms include synthetic gene circuits, yeast mating pathway, signal-to-noise ratio, metabolic control analysis, translational repression, and predictive modelling.

How does the IRP PEST tag improve the circuit?

The PEST degradation tag significantly reduces the half-life of the IRP repressor, allowing the circuit to "tune" its sensitivity and amplitude, facilitating a faster de-repression and response to external stimuli.

Why was the pheromone pathway chosen for this circuit?

The yeast pheromone response pathway was selected because it is a well-characterized "signal processing module" that provides discrete, quantifiable, and modifiable behaviours suitable for testing synthetic gene circuits.