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Developing a Tri-Stable Toggle Switch: Insights from Brown University's 2006-2007 iGEM Project

This project investigates the development of a tri-stable toggle switch, extending the seminal work by Gardner et al. on E. coli. The goal is to design a switch with three stable states that correspond to distinct gene expressions, allowing for versatile genetic manipulation. Key objectives include modeling system evolution based on kinetics, extracting necessary parameters through experiments, and demonstrating the designed system. Despite challenges in completing ligation, the project laid foundational work in genetic architecture and simulation models.

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Developing a Tri-Stable Toggle Switch: Insights from Brown University's 2006-2007 iGEM Project

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  1. On Developing a Tri-stable Toggle Switch An investigation into Brown University's 2006-2007 IGEM project George Washington

  2. 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

  3. 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

  4. The Design

  5. The Players • AraC represses the pAraC/BAD promoter • L-arabinose inactivates AraC, allowing transcription • AraC forms a dimer structure when repressing

  6. 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

  7. The Players • TetR represses the pTet promotor • Tetracycline inactivates TetR, but anhydrotetracycline is used here • TetR naturally forms a dimer structure

  8. 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

  9. The Model

  10. The Model (simplified) • i =rate of production by promoter i • i = cooperativity of repressor i

  11. 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

  12. 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

  13. 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 

  14. 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

  15. 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

  16. 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

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