1. GENETIC TOGGLE SWITCH IN E. COLI COLLINS LAB (2000) Allen Lin 1

2. Main Idea  Bistable switch  Repressor inhibits transcription, inducer promotes transcription (by inhibiting repressor) 2

3. Toggle Model  u = [repressor 1]  v = [repressor 2]  alpha1 = effect rate of synthesis of repressor 1  alpha2 = effect rate of synthesis of repressor 2  beta = cooperatively of repression of promoter 2  gamma = cooperatively of repression of promoter 1 3  Sigmodal shape from cooperative repression of transcription greater than 1  Rates of synthesis of two repressors needs to be balanced

4. Plasmid  pTAK class  Lac repressor (LacI) with Ptrc-2 promoter  Temp-sensitive Lambda repressor (cIts) with PLs1con promoter  Switched by IPTG or thermal pluse  pIKE class  Lac repressor (LacI) with Ptrc-2 promoter  Tet repressor (tetR) with PLtetO-1 promoter  Switched by IPTG or aTc  Transcriptional efficiencies: PLs1con > Ptrc-2 > PLtetO-1  Rates of synthesis of repressors changed by modifying RBS (RBS1 in particular) 4

5. GFP  Gpmut3 gene downstream of Ptrc-promoter  Transcription of Ptrc-2 >> GFP expressed (high)  Transcription of P1 >> GFP not expressed (low) 5

6. Bistability in 5 plasmids  5 bistable toggle plasmids  pIKE differ from expected  Tet repressor (in pIKE) weaker than lambda repressor (in pTAK). PtetO- 1 promoter in pIKE105 not reduced enough 6

7. Long term bistability  pTAK117 7

8. Threshold  After 17-h induction  pTAK117 (red) exhibits quasi- jump  pTAK102 (blue) control exhibits sigmoidal curve  3a and 3b high and low of bimodally distributed curves  Figure b - w/o gene expression variability 8

9. Blur at Threshold 9

10. Switching Time  Switching time depends on rate of elimination of repressor protein  Low to high – dilution by cell growth of IPTG-bound Lac repressor  High to low – thermal destabilization of temp- sensitive lambda repressor  Faster to switch to low state than to high state 10