On Developing a Tri-stable Toggle Switch
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On Developing a Tri-stable Toggle Switch An investigation into Brown University's 2006- 2007 IGEM project George Washington
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On Developing a Tri-stable Toggle Switch. An investigation into Brown University's 2006-2007 IGEM project. George Washington. Goals. Design a switch with three stable states corresponding to three different gene expressions Be able to model the evolution of the system from its base kinetics - PowerPoint PPT Presentation
Transcript of On Developing a Tri-stable Toggle Switch
On Developing a Tri-stable Toggle Switch
An investigation into Brown University's 2006-2007 IGEM project
George Washington
Goals
● Design a switch with three stable states corresponding to three different gene expressions
● Be able to model the evolution of the system from its base kinetics
● Develop and carry out experiments that will extract the parameters for the model
● Build and demonstrate the system
Why?
● In 2000, Gardner et al. developed a toggle switch in E-coli with two stable states
● The ability to set a genetic system into one of multiple stable states is invaluable
● Brown's work is a natural extension of Gardner's
The Design
The Players
● AraC represses the pAraC/BAD promoter● L-arabinose inactivates AraC, allowing
transcription● AraC forms a dimer structure when repressing
The Players
● LacI represses the pLac promoter● Lactose inactivates LacI, although in this case, the
equivalent IPTG is used● LacI naturally forms a tetramer structure
The Players
● TetR represses the pTet promotor● Tetracycline inactivates TetR, but
anhydrotetracycline is used here● TetR naturally forms a dimer structure
The Model
● Some reactions are relatively fast and reversible Formation of multimers from monomer components Binding of repressors to promoter regions
● Others are much slower and irreversible Gene expression Protein degradation
● This distinction gives a basis for a continuous model of system evolution in time
The Model
The Model (simplified)
● i =rate of production by promoter i● i = cooperativity of repressor i
Model Results
● A strong dependence on of system stability was determined
● At high values, small perturbations in repressor concentration are unlikely to influence the system
● For less than one, tristability disappears
Establishing Parameters
● To measure , a simple reporter system would be established
● Production of GFP after introduction of a ligand would indicate overall production due to the promoter
● The strength of the RBS could be modified to achieve values of needed for tristability
Establishing Parameters
● To measure , a slightly more complex system was devised
● Inducing the first promoter makes GFP concentration match the repressor's concentration, so GFP vs YFP will give
Establishing Parameters
● Inducer concentration should be optimized such that an overabundance of ligand is avoided
● In this test, one simply measures GFP vs Inducer concentration to extract optimal levels
Results of the Project
● Designed the genetic architecture required
● Derived the models to be used for simulation of the system
● Designed the tests to be used to establish parameters
● Weren't able to finish ligation, so testing couldn't yet begin
References
● Brown University's IGEM presentation and website http://parts.mit.edu/igem07/index.php/Tristable
● Gardner, T.S., Cantor, C.R., and Collins, J.J.: ‘Construction of a genetic toggle switch in Escherichia coli’, Nature, 2000, 304, pp. 339–342
