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Genetic Toggle Switch in E. Coli Collins Lab (2000)

Genetic Toggle Switch in E. Coli Collins Lab (2000). Allen Lin. Main Idea. Bistable switch Repressor inhibits transcription, inducer promotes transcription (by inhibiting repressor). Toggle Model. u = [repressor 1] v = [repressor 2] alpha1 = effect rate of synthesis of repressor 1

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Genetic Toggle Switch in E. Coli Collins Lab (2000)

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  1. Genetic Toggle Switch in E. ColiCollins Lab (2000) Allen Lin

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

  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 • 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)

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

  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

  7. Long term bistability • pTAK117

  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

  9. Blur at Threshold

  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

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