Construction of a genetic toggleswitch in Escherichia coli

Farah and Tom

What is a Toggle Switch?

“A synthetic bistable gene regulatory network”

What is a Toggle Switch?

The switch consists of:

Promoters

Repressors

Inducers

Promoters encourage expression of a gene.

Repressors bind to promoters, inhibiting expression of genes.

Inducers bind to repressors, preventing repressor binding to promoters and thus encouraging expression.

These components are arranged in a mutually inhibitory fashion.

What is a Toggle Switch?

This model was chosen as it was:

- Simple

- Achieves robust bistable behaviour

How does the toggle switch work?

Promoter 1 encourages expression of Repressor 2

Promoter 2 encourages expression of Repressor 1

Assuming Repressor 2 is in excess of Repressor 1

• Promoter 1 is not active

• On introduction of Inducer to Repressor 2

• Inducer binds repressor 2

• Repressor 2 can no longer bind Promoter 2

• Promoter 2 promotes expression of Promoter 1

• Switching has occurred

The Maths

vudt

dv

1

2

uvdt

du

1

1

The Geometric Structure of the Toggle Equations

pTAK and pIKETwo classes of toggle switches were constructed:

pTAK

lac repressor represses the Ptrc-2 promoter

Temperature sensitive lambda repressor represses the PLslcon promoter

Induced by: IPTG (lac) or Thermal Pulse (tsLambda)

pIKE

lac repressor represses the Ptrc-2 promoter

tet repressor represses PLtetO-1

Induced by: IPTG (lac) or aTc (tet)

High and Low StatesGreen Fluorescent Protein (GFP) is used as a reporter.

GFP is under control of Ptrc-2

Example of a pIKE system with GFP reporter

The state where Ptrc-2 is active, and GFP is being produced is called the High State

The state where Ptrc-2 is repressed, and GFP is not being produced is called the Low State

LacI GFPPtrc-2 PLtetO-1TetR

Investigating BistabiltySix variants were used to investigate the conditions required for bistability

• Four pTAK plasmids

• Two pIKE plasmids

These variants differed by the strength of Ribosome Binding Site 1 (RBS1), where stronger binding RBS sites lead to greater protein synthesis.

All the pTAK plasmids exhibited bistability and

remained stable for at least 22h.

One of the pIKE plasmids exhibited bistability

The reason for the failure of this pIKE is most likely

because TetR is a weaker repressor than lambda.

Graphs

Switching StatesFor pTAK117 switching time for low to high state takes around six hours.

Switching StatesFor pTAK117 switching time for high to low state takes around 30 minutes.

Switching StatesSwitching from Low to High

This takes upto 6 hours.

This is because IPTG-bound Lac Repressor is gradually diluted by cell growth.

As IPTG-bound Lac Repressor decreases, the amount of TetR produced also decreases, allowing expression of LacI and GFP

Switching from High to Low

This takes upto 35 minutes.

This is because tsLamdba is immediately destabilised by a temperature increase

LacI GFPPtrc-2 PLtetO-1TetR

Other potential togglesIn the examples of toggle switches used here the mechanism used to flip the switch has been introduction of an inducer.

This has limited application in synthetic biology.

There are other ways of achieving the switch which may be of use:

- Addition of extra repressor

- Destruction of repressor (eg. Using UV light)

Applications

Gene Therapy

Biotechnology

Cellular memory unit