Genetic Toggle Switch construction and modeling Toggle switch

Genetic Toggle Switch construction and modeling

Toggle switch design

How it works? State 0 X X

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

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

Gene Cloning

Toggle switch plasmid How to select promoters?

Model - Equations x – concentration of repressor 1 y – concentration of repressor 2 a 1, 2 – effective rate of synthesis of x, y b, g – cooperativity of repression from promoter 1, 2

Results Nullclines stable steady state (High) y unstable steady state x Parameters a 1=a 2=100 b=g=2 (b, g>1) stable steady state (Low) • three steady states; one unstable, two stable – bistability • in the case of b, g=1 there is one stable steady state

Results Bifurcation diagram Bistability requires: • a 1, a 2 do not differ greatly • b, g are not drastically different • how “drastic” difference of a 1 vs. a 2 is tolerated depends on the magnitude of b and g

Estimation of a 1 and a 2 Recombinant promoters used: • PL-s 1 con>Ptrc-2>PLtet. O-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

Bistable constructs construct P 1 RBS 1 p. TAK 117 PLs 1 con D p. TAK 130 PLs 1 con G p. TAK 131 PLs 1 con F p. TAK 132 PLs 1 con H p. IKE 107 PLtet. O-1 C R 1 is c. Its for p. TAK series tet. R for p. IKE series

Demonstration of bistability

Model predictions - treshold p. TAK 117 a 1=156. 25 a 2=15. 6 b=2. 5 g=1 m=2. 0015 K=2. 9818 E-5 3. 92323 e-5 Loc. Bif/Win. PP

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

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