On Developing a Tri-stable Toggle
Switch
An investigation into Brown University's 2006-2007 IGEM project
George Washington
Goals
●
●
●
●
Design a switch with three stable states
corresponding to three different gene expressions
Be able to model the evolution of the system from
its base kinetics
Develop and carry out experiments that will
extract the parameters for the model
Build and demonstrate the system
Why?
●
●
●
In 2000, Gardner et al.
developed a toggle switch
in E-coli with two stable
states
The ability to set a genetic
system into one of
multiple stable states is
invaluable
Brown's work is a natural
extension of Gardner's
The Design
The Players
●
●
●
AraC represses the pAraC/BAD promoter
L-arabinose inactivates AraC, allowing
transcription
AraC forms a dimer structure when repressing
The Players
●
●
●
LacI represses the pLac promoter
Lactose inactivates LacI, although in this case, the
equivalent IPTG is used
LacI naturally forms a tetramer structure
The Players
●
●
●
TetR represses the pTet promotor
Tetracycline inactivates TetR, but
anhydrotetracycline is used here
TetR naturally forms a dimer structure
The Model
●
Some reactions are relatively fast and reversible


●
Others are much slower and irreversible


●
Formation of multimers from monomer components
Binding of repressors to promoter regions
Gene expression
Protein degradation
This distinction gives a basis for a continuous
model of system evolution in time
The Model
The Model (simplified)
●
●
i =rate of production by promoter i
i = cooperativity of repressor i
Model Results
●
●
●
A strong dependence
on  of system stability
was determined
At high  values, small
perturbations in
repressor concentration
are unlikely to
influence the system
For  less than one,
tristability disappears
Establishing Parameters
●
●
●
To measure , a simple
reporter system would be
established
Production of GFP after
introduction of a ligand
would indicate overall
production due to the
promoter
The strength of the RBS
could be modified to achieve
values of  needed for
tristability
Establishing Parameters
●
●
To measure , a
slightly more complex
system was devised
Inducing the first
promoter makes GFP
concentration match
the repressor's
concentration, so GFP
vs YFP will give 
Establishing Parameters
●
●
Inducer concentration
should be optimized
such that an
overabundance of
ligand is avoided
In this test, one simply
measures GFP vs
Inducer concentration
to extract optimal
levels
Results of the Project
●
●
●
●
Designed the genetic
architecture required
Derived the models to
be used for simulation
of the system
Designed the tests to
be used to establish
parameters
Weren't able to finish
ligation, so testing
couldn't yet begin
References
●
●
Brown University's IGEM presentation and
website
http://parts.mit.edu/igem07/index.php/Tristable
Gardner, T.S., Cantor, C.R., and Collins, J.J.:
‘Construction of a genetic toggle switch in
Escherichia coli’, Nature, 2000, 304, pp. 339–342