BioSketch

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Back to IGEM_2005

Contents

Goal

  • The idea of the "BactoSketch" (new names are welcome) is that you can wave a "pen" over a lawn or film of bacteria, and those cells that have been stimulated would respond by producing a detectable signal (e.g. luminescence or fluorescence). The cells would remember their state until they receive a second signal that erases the pad.
  • Extras could include multiple output colors or gradation of expression.

Names

  • Bacto-Sketch
  • BioSketch
  • Bacto-Doodle
  • Sketch-a-Doodle
  • BioDoodle
  • Others?

Circuit Design

Image:BactoSketchDiagram_2005-06-06.jpg

  • Pen: UV light
    • 1-10s of UV transiently inhibits A
  • Memory: toggle switch
    • inhibition of A permits B to be synthesized
    • high B concentration is maintained by B inhibiting A
  • Ink: luminescence/fluorescence
    • inhibition of A permits the synthesis of reporter protein
    • this could also be regulated at the translational/mRNA level
  • Eraser: temperature
    • high temperature transiently inhibits B
    • inhibition of B enables A to be dominate again, inhibiting reporter

BioSketch Components

Construction Phases

Temperature as input (Coaster) - Regulate reporter(s) at high (or low) temperature

  • Heat --> Reporter
  • Heat --| Reporter
  • Heat --| B --> Reporter

UV as input - Regulate reporter after brief exposure to UV

  • UV --> Reporter
  • UV --| A --> Reporter
  • UV --| A --| Reporter

Latch - Express reporter in response to transient UV and maintain

  • UV --| A --| Reporter, UV --| A --| B --| A
  • Only worry about switching from A to B at this point

Toggle-switch

  • Full circuit complete - switch between A and B
  • Switch to B state after UV exposure: UV --| A --| Reporter, UV --| A --| B --| A
  • Switch to A state after heat: Heat --| B --| A --| Reporter, Heat --| B --| A --| B

Molecular Switches

LacIts

For both Collins and BioBricks, LacI will have to be mutated to be temperature sensitive.

From Hasan & Seybakki: Gly187->Ser (G(568)GC->AGC)
From Yabuta (better?): Ala241->Thr (G(730)CG->ACG)


Collins CI-LacIts Switch


What we got from Collins Collins has FedEx'ed us on 14 June 2005 the 6 plasmids that we requested (on 13 June 2005). The plasmids were pCIRa, pCIRb, pCIE, pTSMa, pTSMb1, and pTSMb2 from Kobayashi et al., 2004 (PNAS 2004). They arrived on 15 June 2005, but we had to order antibiotics which arrived on 16 June 2005.

Collins also emailed us with the sequences for pTSMb1 and pCIE as well as an MS Excel file with construct descriptions: Collins Constructs.

Note: JM 2.300 is CGSC #5002: [1] MC4100 [2]

Collins emailed us the rest of the sequences on 16 June 2005.

Sequences:


Details of the switch

  • Writing via Repression
    • Reporters
      • pWG[P(L)*_gfpmut3b]: pCIRa
      • pWC[P(L)*_mCherry]: pCIRa, gfp replaced w/ mCherry
    • OFF Switch
      • pWCI[P(trc)_cI]: pCIE
    • Experiments
      • pWG (+ ctrl for signal)
      • pWC (+ ctrl for signal)
      • pWCI + pWG (UV-induced)
      • pWCI + pWC (UV-induced)


  • Erasing via Repression
    • Reporters
      • pEG[P(trc)_gfpmut3b]: pCIRa, P(L)* replaced w/ P(trc)
      • pEC[P(trc)_mCherry]: pEG, gfpmut3b replaced w/ mCherry
    • ON Switches
      • pEL[P(L)*_lacI]: pTSMa, del P(trc)_cI
      • pELts[P(L)*_lacIts]: pEL, ts mut in lacI
    • Experiments
      • pEG (+ ctrl for signal)
      • pEC (+ ctrl for signal)
      • pEL + pEG (IPTG-induced; + ctrl for IPTG induction; - ctrl for temp)
      • pEL + pEC (IPTG-induced; + ctrl for IPTG induction; - ctrl for temp)
      • pELts + pEG (heat & IPTG-induced)
      • pELts + pEC (heat & IPTG-induced)


  • Erasing via Induction
    • Reporters
      • pEGhs[P(hs)_gfpmut3b]: pCIRa, P(L)* replaced w/ P(hs), heat-shock promoter
      • pWG: GFP reporter from Writing via Repression circuit
      • pWC: mCherry reporter from Writing via Repression circuit
    • OFF Switch
      • pECIhs[P(hs)_cI]: pCIE, P(trc) replaced w/ P(hs)
    • Experiments
      • pEGhs (heat-induced; + ctrl for heat induction)
      • pECIhs + pWG (heat-repressed)
      • pECIhs + pWC (heat-repressed)


  • Toggle Switch
    • Reporters
      • pTSGC[gfpmut3b_P(trc)_-_P(L)*_mCherry]: pEG + pWC, opposite orientations
    • Switches
      • pTS[cI_P(trc)_-_P(L)*_lacI]: pTSMa
      • pTSts[cI_P(trc)_-_P(L)*_lacIts]: pTSMa, ts mut in lacI
      • pECIhs: heat-inducible CI from Erasing via Induction circuit
    • Experiments
      • pTSGC (+ ctrl for signal, both)
      • pTS + pTSGC (Collins switch)
      • pTSts + pTSGC (UV-inducible Cherry, IPTG/heat-inducible GFP)
      • pTS + pECIhs + pTSGC (qualitatively same as pTSts + pTSGC)
      • pTSts + pECIhs + pTSGC (qualitatively same as pTSts + pTSGC)

BioSketch BioBricks

BioSketch BioBricks

BioSketch BioBricks Numbering

BioSketch BioBricks Reporters

BioBrick CI-LacIts Switch

  • First switch: [P(L)]-[RBS-LacI-T-P(Lac)]-[RBS-cI-T-P(L)]-[RBS-GFP-T]
    • First assembly: Join P(L) R0051 with LacI inverter Q04121; Join lambda cI inverter Q04510 with GFP reporter I13504
    • Second assembly: Join [P(L)]-[LacI inverter] with [cI inverter]-[GFP reporter]
  • Later:
    • mutate LacI -> LacI(ts)
    • Replace [RBS-GFP-T] with new [RBS-mCherry-T] component
    • Add reporter driven by LacI promoter (report off state)
    • Separate high-copy reporter plasmid if reporting needs augmenting

BioBrick 434-CI587 Switch

Experiments

Records

Presentations


BioSketch References