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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. - PowerPoint PPT Presentation
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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