1. Construction of a genetic toggle switch in Escherichia coli Farah and Tom

2. What is a Toggle Switch? “A synthetic bistable gene regulatory network”

3. 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.

4. What is a Toggle Switch? This model was chosen as it was: - Simple - Achieves robust bistable behaviour

5. 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

6. The Maths

7. The Geometric Structure of the Toggle Equations

8. pTAK and pIKE Two classes of toggle switches were constructed: pTAK lac repressor represses the Ptrc-2 promoter Temperature sensitive lambda repressor represses the P L slcon promoter Induced by: IPTG (lac) or Thermal Pulse (tsLambda) pIKE lac repressor represses the Ptrc-2 promoter tet repressor represses P L tetO-1 Induced by: IPTG (lac) or aTc (tet)

9. High and Low States Green 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 LacIGFPPtrc-2P L tetO-1TetR

10. Investigating Bistabilty Six 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.

11. Graphs

12. Switching States For pTAK117 switching time for low to high state takes around six hours.

13. Switching States For pTAK117 switching time for high to low state takes around 30 minutes.

14. Switching States Switching 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 LacIGFPPtrc-2P L tetO-1TetR

15. Other potential toggles In 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)

16. Applications Gene Therapy Biotechnology Cellular memory unit