Genetic Toggle Switchconstruction and modeling

Toggle switch designToggle switch design

How it works?How it works?

X X

State 0

How it works?How it works?

Switching from state 0 to 1 : applying the inducer 2

How it works?How it works?

X

State 1

To switch back to state 0…

Gene CloningGene Cloning

Toggle switch plasmidToggle switch plasmid

How to select promoters?

Model - EquationsModel - Equations

x – concentration of repressor 1y – concentration of repressor 21,2 – effective rate of synthesis of x,y, – cooperativity of repression from promoter 1,2€

dx

dt=α 1

1+ y β− x

dy

dt=α 2

1+ xγ− y

ResultsResults

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

yunstable

steady state

stablesteady state

(Low)

stablesteady state

(High)

ResultsResults

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

Estimation of 1 and 2Estimation of 1 and 2

Recombinant promoters used:

• PL-s1con>Ptrc-2>PLtetO-1

Mutant RBS sequences (SD):

• A>B>C>D>E>F>G

Overall synthesis strength is P+RBS

• steady state GFP levels measuredthrough fluorescence

Bistable constructsBistable constructs

construct P1 RBS1

pTAK117 PLs1con D

pTAK130 PLs1con G

pTAK131 PLs1con F

pTAK132 PLs1con H

pIKE107 PLtetO-1 C

R1 is cIts for pTAK series tetR for pIKE series

Demonstration of bistabilityDemonstration of bistability

Model predictions - tresholdModel predictions - treshold

pTAK117

1=156.25

2=15.6=2.5=1=2.0015K=2.9818E-5

3.92323e-5

LocBif/WinPP

€

x ⇒x

1+[IPTG]

K

⎛

⎝ ⎜

⎞

⎠ ⎟μ

Genetic circuit analysis and engineeringGenetic circuit analysis and engineering

analyzing existing genetic circuits

construction of new genetic circuits

MODELING

ReferencesReferences

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/readme.htm