1. Genetic Toggle Switch construction and modeling

2. Toggle switch design

3. How it works? XX State 0

4. How it works? Switching from state 0 to 1 : applying the inducer 2

5. How it works? X State 1 To switch back to state 0…

6. Gene Cloning

7. Toggle switch plasmid How to select promoters?

8. Model - Equations x – concentration of repressor 1 y – concentration of repressor 2  1,2 – effective rate of synthesis of x,y , – cooperativity of repression from promoter 1,2

9. Results Parameters  1 = 2 =100 ==2 ( three steady states; one unstable, two stable – bistability in the case of  there is one stable steady state Nullclines x y unstable steady state stable steady state (Low) stable steady state (High)

10. Results Bistability requires:  1,  2 do not differ greatly , are not drastically different how “drastic” difference of  1 vs.  2 is tolerated depends on the magnitude of  and  Bifurcation diagram

11. Estimation of  1 and  2 Recombinant promoters used: PL-s1con>Ptrc-2>P L tetO-1 Mutant RBS sequences (SD): A>B>C>D>E>F>G Overall synthesis strength is P+RBS steady state GFP levels measured through fluorescence

12. Bistable constructs constructP1RBS1 pTAK117P L s1conD pTAK130P L s1conG pTAK131P L s1conF pTAK132P L s1conH pIKE107P L tetO-1C R1 is cIts for pTAK series tetR for pIKE series

13. Demonstration of bistability

14. Model predictions - treshold pTAK117  1 =156.25  2 =15.6 =2.5 =1 =2.0015 K=2.9818E-5 3.92323e-5 LocBif/WinPP

15. Genetic circuit analysis and engineering analyzing existing genetic circuits construction of new genetic circuits MODELING

16. References Gardner TS, Cantor CR, Collins JJ (2000) Construction of a genetic toggle switch in Escherichia coli. Nature 403:339-342 Yagil G, Yagil E (1971) On the relation between effector concentration and the rate of induced enzyme synthesis. Biophys J 11:11-27 WINPP and LOCBIF http://www.math.pitt.edu/~bard/classes/wppdoc/re adme.htm