On Developing a Tri-stable Toggle Switch

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