Team:Aberdeen Scotland

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<h1>Why ayeSwitch?</h1>
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Over the course of the summer, the University of Aberdeen iGEM team engineered a novel genetic toggle switch in yeast which is regulated at the translational level and allows mutually exclusive expression of either green or cyan fluorescent protein. Using cell cytometry (FACS) and fluorimetry, we successfully demonstrated gene expression and translational regulation of a fusion of mRNA binding proteins and fluorescent proteins. Deterministic and stochastic models including experimental results and published parameter values predicted that the probability of successful bistability for the switch is 0.96%, but that this can theoretically be improved to a maximum of 51.27% by limiting the variation range of the most sensitive parameters. The models also predicted that to generate switch-like behaviour, co-operative binding of the mRNA binding protein to its mRNA stem loop was essential.  These results suggest that a translationally regulated genetic toggle switch is a viable and novel engineering concept applicable to medicinal, environmental and technological problems.
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<a href="https://2010.igem.org/Team:Aberdeen_Scotland/Project_Overview">Your ayeSwitch experience begins here&nbsp;<img src="https://static.igem.org/mediawiki/2010/3/36/Right_arrow.png"></a>
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Bio-traffic light systems; engineering toggle switches in yeast using translational control of gene expression
 
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Abstract
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<h1>Our Sponsors:</h1>
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This synthetic biology project will engineer a novel gene circuit allowing yeast to express either a green, or cyan fluorescent protein, in a stable, mutually exclusive way, thus exhibiting toggle switch control of expression. Exposure of yeast to specific environmental signals will switch between the fluorescent proteins. In contrast to existing genetic toggle switches, employing transcriptional control to achieve regulation, this project will achieve the same control principle by engineering a translational regulation circuit in a model eukaryote. A key advantage of translational control is that it enables a more rapid regulation of protein levels since it by-passes mRNA synthesis and export. Translationally regulated toggle switches could have multiple applications, stably recording exposure to environmental triggers, or in long-term maintenance of therapeutic protein synthesis (3).
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Aberdeen iGEM 2010 gratefully acknowledges the financial support of the following organisations:
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We will implement a translational control toggle switch using two proteins, Iron Response element binding Protein (IRP), and MS2-stem loop binding protein (MS2-BP); both proteins bind RNA stem loops in a sequence-specific manner. In the toggle switch circuit, the gene for IRP will contain an MS2 RNA stem in its 5’ leader; a second gene will encode MS2-BP, with an iron response element in its 5’ leader. Each RNA-binding protein can potentially inhibit the translation of the other (1,2; Figure 1). The two RNA binding proteins will be fused to GFP and CFP respectively, to allow fluorescence monitoring of toggle switch position. By additionally regulating the transcription of each RNA-binding protein gene, using galactose- and copper-inducible promoters respectively, a re-set function is established, allowing cyan/green toggling. The project has scope for development if time allows; translational control of stop codon readthrough using suppressor tRNAs can be used to tag IRP-GFP with a destabilising peptide tag, acting as a translationally controlled toggle reset.  
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Mathematical modelling will be used to describe the expression of the circuit’s genes, including both transcription and translation. Initially, we will use ordinary differential equations to predict the concentration of proteins in response to signal input. Later refinement will employ a stochastic model of translation that considers the traffic of ribosomes on the mRNAs, and links to the transcription process. The presence of a protein blocking the 5’ leader of an mRNA will be modelled by reducing the probability with which ribosomes bind the mRNA. Stochastic simulations will moreover allow generalising predictions from single cells to populations of cells. The proposed model will be validated by comparing the predictions to experimental data.
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Overall, the project will test the hypothesis that synthetic toggle switches can be engineered using translational control. The project will additionally establish a translational control toolkit for eukaryote bioengineering.
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The specific objectives and anticipated outcomes of this project are as follows;
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1. Development of deterministic and stochastic mathematical models of translational toggle switches to guide circuit design.
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2. Construction of RNA binding protein BioBricks, fused to GFP and RFP, in yeast expression cassettes.
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3. Testing of RNA-binding protein/fluorescent protein expression in yeast, at the single cell and population levels.
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4. Iterative improvement of the model using experimental data, and test of new predictions generated by the model.
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!align="center"|[[Team:Aberdeen_Scotland|Home]]
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!align="center"|[https://igem.org/Team.cgi?year=2010&team_name=Aberdeen_Scotland Official Team Profile]
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Latest revision as of 19:31, 27 October 2010

University of Aberdeen - ayeSwitch - iGEM 2010

Why ayeSwitch?

Over the course of the summer, the University of Aberdeen iGEM team engineered a novel genetic toggle switch in yeast which is regulated at the translational level and allows mutually exclusive expression of either green or cyan fluorescent protein. Using cell cytometry (FACS) and fluorimetry, we successfully demonstrated gene expression and translational regulation of a fusion of mRNA binding proteins and fluorescent proteins. Deterministic and stochastic models including experimental results and published parameter values predicted that the probability of successful bistability for the switch is 0.96%, but that this can theoretically be improved to a maximum of 51.27% by limiting the variation range of the most sensitive parameters. The models also predicted that to generate switch-like behaviour, co-operative binding of the mRNA binding protein to its mRNA stem loop was essential. These results suggest that a translationally regulated genetic toggle switch is a viable and novel engineering concept applicable to medicinal, environmental and technological problems.




Your ayeSwitch experience begins here 

Our Sponsors:

Aberdeen iGEM 2010 gratefully acknowledges the financial support of the following organisations:





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