On Developing a Tri-stable Toggle Switch

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On Developing a Tri-stable Toggle Switch
An investigation into Brown University's
2006-2007 IGEM project
George Washington
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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

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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

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The Design
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The Players

• AraC represses the pAraC/BAD promoter
• L-arabinose inactivates AraC, allowing
  transcription
• AraC forms a dimer structure when repressing

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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

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The Players

• TetR represses the pTet promotor
• Tetracycline inactivates TetR, but
  anhydrotetracycline is used here
• TetR naturally forms a dimer structure

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The Model

• Some reactions are relatively fast and reversible
• Formation of multimers from monomer components
• Binding of repressors to promoter regions
• Others are much slower and irreversible
• Gene expression
• Protein degradation
• This distinction gives a basis for a continuous
  model of system evolution in time

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The Model
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The Model (simplified)

• ?i rate of production by promoter i
• ?i cooperativity of repressor i

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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

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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

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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 ?

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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

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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

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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. 339342