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