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MURI Pattern Detection Circuits

MURI Pattern Detection Circuits. Andrew Yang, Jonathan Babb, Ron Weiss 9/23/2011. Null versus 1 Systems (Circuits #1-6).

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MURI Pattern Detection Circuits

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  1. MURI Pattern Detection Circuits Andrew Yang, Jonathan Babb, Ron Weiss 9/23/2011

  2. Null versus 1 Systems (Circuits #1-6) NOTE: Not depicted is a stretch of DNA following the promoters and preceding the genes called the ‘RBS’. The RBS is tunable, thus controlling the strength of gene expression. Please account for this possibility. • Circuit operation: Initial state of toggle set for CI production with IPTG. Later, UV induces state change for a region by cleaving CI. If the region is large enough to achieve an engineering quorum, the quorum is amplified to the entire region and reported by a blue fluorescent protein. Goal: Digital behavior. Circle sizes willbe smaller or larger than aforbidden band. UV Toggle 3OC12HSL Quorum Sensor 3OC12HSL Propagator State “1” = 30C12HSL Null Null “1”

  3. Circuit #1 Las System 30C12HSL 30C12HSL UV IPTG LasR GFP LasI LacI pCI pLac CI pLasORI LasI LasR mCherry pLas Toggle Switch Quorum Sensor Propagator Quorum sensor activated only in UV exposed region.

  4. Circuit #2: External atc->LasR atc 30C12HSL 30C12HSL UV IPTG ptet LasR LasI LacI pCI pLac CI pLasORI LasI LasR mCherry GFP pLas Toggle Switch Quorum Sensor Propagator pLacIq TetR Quorum sensor activated only in UV exposed region.

  5. Circuit #3: atc->LasI Activator 30C12HSL pLas Activator atc 30C12HSL UV IPTG pLacIq TetR ptet LasR LasI LacI pCI pLac CI pLasORI LasI LasR mCherry GFP pActivator Toggle Switch Quorum Sensor Propagator Quorum sensor activated only in UV exposed region.

  6. Circuit #4: Const. Lactonaseatc->LasR atc 30C12HSL UV IPTG 30C12HSL ptet LasR LasI AiiA LacI pCI pLac CI pLasORI LasI LasR mCherry GFP pConstitutive pLas Toggle Switch Quorum Sensor Propagator pLacIq TetR Quorum sensor activated only in UV exposed region.

  7. Circuit #4A: Const. LactonaseNo atc AiiA 30C12HSL UV IPTG 30C12HSL LasI LacI pCI pLac CI pLasORI LasI LasR mCherry GFP pLas Toggle Switch Quorum Sensor Propagator pLacIq LasR Quorum sensor activated only in UV exposed region.

  8. Circuit #5: atc->LactonasePositive Feedback LasI ptet AiiA atc 30C12HSL 30C12HSL UV IPTG LasR LasI LacI pCI pLac CI pLasORI LasI LasR mCherry GFP pLas Toggle Switch Quorum Sensor Propagator pLacIq TetR Quorum sensor activated only in UV exposed region.

  9. Circuit #6: atc->Inducible LactonasePositive Feedback LasI pLas/tetO AiiA atc 30C12HSL UV IPTG 30C12HSL LasR LasI LacI pCI pLac CI pLasORI LasI LasR mCherry GFP pLas Toggle Switch Quorum Sensor Propagator pLacIq TetR Quorum sensor activated only in UV exposed region.

  10. Null versus 1 versus 2 Systems (Circuit #7) NOTE: As before, not depicted is a stretch of DNA following the promoters and preceding the genes called the ‘RBS’. The RBS is tunable, thus controlling the strength of gene expression. Please account for this possibility. Key: LasR has low sensitivity and only detects large circles (2) while RhiR system has high sensitivity for small circles (1). TetR represses small circles if when large are also present. Null Null “1” “2” • Operations: now we have two separate quorum sensing systems tuned to different sensitivities. To detect large circles, LasR will amplify with a low sensitivity. In the case where there is no large circle, a more sensitive RhiR system will detect and amplify a smaller circle. Forbidden bands in between each range are not unused by the digital input. UV Toggle 3OC12HSL Quorum Sensor 3OC12HSL Propagator C4HSL Quorum Sensor C4HSL Propagator State “1” = C4 ^ C12 State “2” = C12 Null = NOR(C4,C12)

  11. Circuit #7: Null versus 1 versus 2 system C4HSL C4HSL 30C12HSL 30C12HSL UV IPTG RhiR GFP RhiI pRhiORI RhiI RhiR mCherry pRhi LasR BFP LasI LacI pCI pLac CI pLasORI LasI LasR mCherry pLas Toggle Switch Propagator Quorum Sensors

  12. Appendix Following are circuit #1 with the Rhil and Lux systems. The same changes in promoters, inducers, and repressors can be applied to circuits #2-6.

  13. Circuit #1 Rhil System C4HSL C4HSL UV IPTG RhiR GFP RhiI LacI pCI pLac CI pRhiORI RhiI RhiR mCherry pRhi Toggle Switch Quorum Sensor Propagator Quorum sensor activated only in UV exposed region.

  14. Circuit #1 Lux System C6HSL C6HSL UV IPTG LuxR GFP LuxI LacI pCI pLac CI pLuxORI LuxI LuxR mCherry pLux Toggle Switch Quorum Sensor Propagator Quorum sensor activated only in UV exposed region.

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