Genetic Toggle Switch - PowerPoint PPT Presentation

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Genetic Toggle Switch

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x concentration of repressor 1. y concentration of repressor 2 ... tetR for pIKE series. Demonstration of bistability. Model predictions - treshold. pTAK117 ... – PowerPoint PPT presentation

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Title: Genetic Toggle Switch


1
Genetic Toggle Switch
  • construction and modeling

2
Toggle switch design
3
How it works?
State 0
X
X
4
How it works?
Switching from state 0 to 1 applying the
inducer 2
5
How it works?
State 1
X
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 a1,2 effective
rate of synthesis of x,y b,g cooperativity of
repression from promoter 1,2
9
Results
Nullclines
  • Parameters
  • a1a2100
  • bg2 (b, ggt1)
  • three steady states one unstable, two stable
    bistability
  • in the case of b, g1
  • there is one stable steady state

stable steady state (High)
y
stable steady state (Low)
unstable steady state
x
10
Results
Bifurcation diagram
  • Bistability requires
  • a1, a2 do not differ greatly
  • b,g are not drastically different
  • how drastic difference of a1 vs. a2 is
    tolerated depends on the magnitude of b and g

11
Estimation of a1 and a2
  • Recombinant promoters used
  • PL-s1congtPtrc-2gtPLtetO-1
  • Mutant RBS sequences (SD)
  • AgtBgtCgtDgtEgtFgtG
  • Overall synthesis strength is PRBS
  • steady state GFP levels measured
  • through fluorescence

12
Bistable 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
13
Demonstration of bistability
14
Model predictions - treshold
  • pTAK117
  • a1156.25
  • a215.6
  • b2.5
  • g1
  • m2.0015
  • K2.9818E-5

3.92323e-5
LocBif/WinPP
15
Genetic circuit analysis and engineering

analyzing existing genetic circuits
MODELING
construction of new genetic circuits
16
References
  • Gardner TS, Cantor CR, Collins JJ (2000)
    Construction of a genetic toggle switch in
    Escherichia coli. Nature 403339-342
  • Yagil G, Yagil E (1971) On the relation between
    effector concentration and the rate of induced
    enzyme synthesis. Biophys J 1111-27
  • WINPP and LOCBIF http//www.math.pitt.edu/bard/
    classes/wppdoc/readme.htm
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