Principal Supervisor: Prof John McCarthy - School of Life Sciences
Co-supervisor: Prof. Declan Bates - Engineering
PhD project title: A Posttranscriptional Paradigm for Synthetic Biological Circuitry
University of Registration: Warwick
This project will seek to establish a proof-of-concept basis for ground-breaking technology and applications in Industrial Biotechnology. It will achieve this by providing an entirely new alternative to the circuitry based on transcriptional components that is currently used universally in the exciting new research area of Synthetic Biology. Such circuitry is employed in various ways to construct biological systems capable of functions including computation, sensing, production of valuable bioactives, degradation of industrial xenobiotic contaminants and bioenergy production. The limitations of transcriptional components as building blocks for synthetic circuitry include inter-component incompatibility, restricted dynamic range of regulation, and poor kinetics/switching response times. The student working on this project will be able to develop radically new types of fast-acting protein- and RNA- based circuitry that will ultimately find applications relevant to biotechnology, healthcare, the environment, food security and bioenergy. Advanced modelling will be an integrated feature of the project, facilitating system design and data interpretation.
The first phase of this project will capitalise on earlier experimental work in the McCarthy lab on translational regulation. For example, we showed previously that the human Iron Regulatory Protein (IRP) suppresses translation of any target eukaryotic mRNA that contains the Iron Response Element (IRE) in its 5’UTR1. This provides the basis for a translational regulatory system that is analogous to, but faster-acting than, a promoter/transcription factor pair. In order to relieve IRP-mediated repression, and therefore induce expression of the reporter, IRP will be fused to the phosphodegron derived from Tec1, a protein that is subject to regulation by the yeast pheromone signalling pathway. In yeast, binding of α-factor pheromone to its G-protein Coupled Receptor Ste2 activates the pheromone signalling pathway, resulting in a phosphorylation cascade whose final target is the MAP Kinase Fus3. Activated Fus3 phosphorylates Tec1 at a specific Threonine residue within its phosphodegron motif. This targets Tec1 for ubiquitylation by the ubiquitin-ligase SCFCdc4 and subsequent degradation. Fusion of the Tec1 phosphodegron with IRP will therefore create a protein-mediated regulatory switch that directly targets mRNA translation. In the first instance, the phosphodegron-IRP/IRE pair will be used to target a reporter mRNA (encoding, for example, ymNeonGreen), but subsequent circuit designs will incorporate alternative targets that can be integrated within more advanced circuitry. Moreover, chimeric human/yeast G proteins will be used to render the system responsive to pharmaceutically relevant ligands (e.g. somatostatin, serotonin, purinergic nucleotides), thus creating multiple versions of the new regulatory system.
In a parallel stream of activity, the PhD student will be able to collaborate with the group of Victor Sourjik (Max Planck Institute, Marburg) in order to adapt components of the Escherichia coli chemosensory system to function in yeast. Specific methyl-accepting chemotaxis proteins (MCPs) will be selected for integration into a further synthetic kinase cascade that can function independently from the pheromone-signalling-pathway-based system outlined above. The MCP-driven synthetic pathway will link in to a different set of regulatory targets.
Finally, signalling pathways involving phosphorylation cascades have been the subject of extensive research by mathematical modellers in recent years, resulting in a wealth of information being available in the Systems Biology literature on the dynamics, sensitivity and robustness of such systems. Due to the absence of the type of noisy dynamics often associated with transcriptional networks, phosphorylation cascades can typically be represented using standard ordinary differential equation (ODE) or partial differential equation (PDE) based models, thus strongly facilitating their mathematical analysis and reducing computational overheads. Working with the project co-supervisor, the student will adapt existing models of phosphorylation cascades from the literature, parameterise them using data from the proposed experimental work, and develop them so that they can be used as CAD tools for the rational design of synthetic post-transcriptional circuitry.
The project will ultimately generate an entirely novel toolbox of synthetic posttranscriptional components that will be tested and characterised using state-of-the-art quantitative methods.
BBSRC Strategic Research Priority: Molecules, cells and systems
Techniques that will be undertaken during the project:
- Robotics methods for the optimization of system functionality
- Synthetic genetic design and assembly
- Protein analysis including mass spectrometry
- Characterisation of gene expression and regulation using biophysical techniques including flow cytometry
- Computational modelling and design
Contact: Professor John McCarthy, University of Warwick