BioSketch The bacterial sketch pad.

BioSketchBioSketchThe bacterial sketch pad.The bacterial sketch pad.

Chris Doucette, Thomas Noriega, Hing Eng, Yves Wang, Jennifer Gao, Yin Li

Group Meeting2005-06-13

BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005

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The IdeaThe IdeaWrite with UV light.Erase by turning up the heat.Remember with a genetic switch.Play with bacteria!Advance synthetic biology.

What more do you want?


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The Toggle Switch: It The Toggle Switch: It Remembers!Remembers!

ON SwitchON SwitchOFF SwitchOFF Switch


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PenPen

OFF OFF to ON: Writingto ON: Writing

ON SwitchON Switch InkInkOFF SwitchOFF Switch

OFF to OFF to ONON: Writing: Writing


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OFF to ON: Alternative OFF to ON: Alternative SchemesSchemes

ON SwitchON Switch InkInk

PenPen

OFF SwitchOFF Switch


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ONON to OFF: Erasing to OFF: Erasing


EraserEraser


ONON to to OFFOFF: Erasing: Erasing


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ON to OFF: Alternative ON to OFF: Alternative SchemesSchemes


EraserEraser



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The PenThe PenRequired Properties

Rapidly induces response/short exposureTransient/reversibleHigh spatial resolution

CandidatesChemical signals

AHLIPTG

Electromagnetic wavesInfrared (heat)UV radiation (254nm)

DevicesChemical signals:

Microspray

Electromagnetic waves:

LaserMicroscopeLamp & stencil

Kobayashi et al. 2004. Kobayashi et al. 2004. PNASPNAS 101: 8414-8419. 101: 8414-8419.


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The OFF SwitchThe OFF SwitchRequired Properties

Rapidly repressible by PenOR

Inducible by EraserAntagonistic to ON Switch

Candidates434 repressor CI repressorLexA repressor (How well-characterized is this? Also cleaved by RecA; Higashitani et al. (1995) gives induction curves.)

Kobayashi et al. 2004. PNAS 101: 8414-8419; Yasuda et al., 1998. EMBOKobayashi et al. 2004. PNAS 101: 8414-8419; Yasuda et al., 1998. EMBO J 17: 3207-3216; J 17: 3207-3216; Gardner et al., 2000. Nature 403: 339-342; Salles & Paoletti. 1983. PNAS 80: 65-69.Gardner et al., 2000. Nature 403: 339-342; Salles & Paoletti. 1983. PNAS 80: 65-69.


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The The CI Repressor CI RepressorProperties:

Cleaved when bound by RecA activated in SOS response to UV irradiation (rapid response at the protein level)Represses variants of PL promoters

Has been used as component of UV-responsive genetic toggle switches

Kobayashi et al. 2004. PNAS 101: 8414-8419; Yasuda et al., 1998. EMBOKobayashi et al. 2004. PNAS 101: 8414-8419; Yasuda et al., 1998. EMBO J 17: 3207-3216; J 17: 3207-3216; Gardner et al., 2000. Nature 403: 339-342.Gardner et al., 2000. Nature 403: 339-342.


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The 434 repressorThe 434 repressorProperties:

Cleaved when bound by RecA activated in SOS response to UV irradiation (rapid response at the protein level)Several variants exist with altered DNA-binding specificities, and improved affinity.

Suggested by Pam, endorsed by Ira.

Pawlowski et al. 2004. J Bacteriol 186: 1-7; Simoncsits et al. 1999. Genetica 106: 85-92; Simoncsits et al. 1999. Pawlowski et al. 2004. J Bacteriol 186: 1-7; Simoncsits et al. 1999. Genetica 106: 85-92; Simoncsits et al. 1999. Nucleic Acids Res 27: 3474-3480.Nucleic Acids Res 27: 3474-3480.


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Turning On the Switch with Turning On the Switch with UVUV

UVUV

Kobayashi et al. 2004. PNAS 101: 8414-8419; Yasuda et al., 1998. EMBOKobayashi et al. 2004. PNAS 101: 8414-8419; Yasuda et al., 1998. EMBO J 17: 3207-3216;J 17: 3207-3216;Higashitani et al. 1995. J Bacteriol 177: 3610-3612; Salles & Paoletti. 1983. PNAS 80: 65-69; Pawlowski & Higashitani et al. 1995. J Bacteriol 177: 3610-3612; Salles & Paoletti. 1983. PNAS 80: 65-69; Pawlowski & Koudelka. 2004. J Bacteriol 186: 1-7.Koudelka. 2004. J Bacteriol 186: 1-7.

RecARecA

dsDNAdsDNAssDNAssDNA

RecA*RecA*

434434

ONON

oNswitchoNswitch434434OOONONPP

434434ONONOOPPRMRM


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The EraserThe EraserProperties

Effects reversibleEasily controlledLow spatial resolution

CandidatesChemical signalsTemperature/heat


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The ON SwitchThe ON SwitchRequired Properties

Repressible by EraserOR

Rapidly inducible by PenAntagonistic to ON Switch component

CandidatesTemperature-sensitive repressors

LacI(ts) (Hasan & Szybalski. 1995. Gene 163: 35-40; Wang et al. 2004. Biotechnol Prog 20: 1350-1358; Yabuta et al. 1995. J Biotechnol 39: 67-73; Chao et al. 2002. Biotechnol Bioeng 79: 1-8; Bukrinsky et al. 1988. Gene 70: 415-417.)

CI857 (Gardner et al. 2000. Nature 403: 339-342; Jechlinger et al. 1999. FEMS Microbiol Let 173: 347-352; Villverde et al. 1993. App Environ Micrbiol 59: 3485-3487.)

Mu c (Vogel et al. 1991. J Bacteriol 173: 6568–6577.)

Tet (Wissmann et al. 1991. Genetics 128: 225-232.)

AraC (Irr & Englesberg. 1971. J Bacteriol 105: 136-141.)

Thermo-labile mRNAs


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The LacI(ts) RepressorThe LacI(ts) RepressorProperties

Loses capacity to repress at 42CRepresses promoters containing lacOWT lacI has been used in genetic toggle switches, and other synthetic circuits.

Several VariantsSer300Asn (Yabuta et al., 1995)

Gly187Ser (Chao et al., 2002)

Leu233Lys (Chao et al., 2002)

Ala241Thr (Chao et al., 2002; Yabuta et al., 1995)

Gly265Asp (Chao et al., 2002; Yabuta et al., 1995)

Burkrinsky et al. (1988) uses a lacIts variant, but the mutation was not disclosed; this paper is cited by Hasan & Szybalski (1995) and Andrews et al. (1996)

Hasan & Szybalski. 1995. Gene 163: 35-40; Wang et al. 2004. Biotechnol Prog 20: 1350-1358; Yabuta et al. Hasan & Szybalski. 1995. Gene 163: 35-40; Wang et al. 2004. Biotechnol Prog 20: 1350-1358; Yabuta et al. 1995. J Biotechnol 39: 67-73; Chao et al. 2002. Biotechnol Bioeng 79: 1-8; Bukrinsky et al. 1988. Gene 70: 1995. J Biotechnol 39: 67-73; Chao et al. 2002. Biotechnol Bioeng 79: 1-8; Bukrinsky et al. 1988. Gene 70: 415-417.415-417.


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The The CI857 Repressor CI857 RepressorProperties:

Functional at 30 C, nonfunctional at 42 C.Not cleaved in SOS response.Used in the original genetic toggle switch.

Suggested by Pam, endorsed by Ira.

Gardner et al. 2000. Nature 403: 339-342; Jechlinger et al. 1999. FEMS Microbiol Let 173: 347-352; Villverde et Gardner et al. 2000. Nature 403: 339-342; Jechlinger et al. 1999. FEMS Microbiol Let 173: 347-352; Villverde et al. 1993. App Environ Micrbiol 59: 3485-3487; Hoffmann et al. 1999. FEMS Microbiol Let 177: 327-334.al. 1993. App Environ Micrbiol 59: 3485-3487; Hoffmann et al. 1999. FEMS Microbiol Let 177: 327-334.


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Turning Off the Switch with Turning Off the Switch with HeatHeat

CI857CI857CI857CI857--

434434

434434cIcIOOPPRMRM cI857cI857434434OOPPRMRM


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The InkThe InkProperties

Repressed by OFF switchOR

Induced by ON switchRapidly inducible (preferred)Rapidly degraded (preferred)Reversible

CandidatesmCherry: monomeric dsRed derivative, rapid folding, shows up purple in visible lightVenus: rapidly-folding (~2 min) YFP variantCa2+-sensitive GFPcAMP-sensitive GFP


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mCherrymCherryRapid folding

Monomeric, as opposed to tetrameric dsRedDeveloped by directed evolution - selection for maturation speed and brightness50% of maximum fluorescence 15 min. post-translationExcited by visible light

Visible without microscopy

Advantageous - UV excitation could interfere with drawingMore immediate results for user

Shaner et al. 2004 Nature Biotechnol 22: 1567-1572.Shaner et al. 2004 Nature Biotechnol 22: 1567-1572.


2020

Venus YFPVenus YFPExtremely rapid folding

Measurable results in less than 2 minutes post-translation (Kirsten Frieda, Harvard REU 2003)

Convenient for early test constructs to estimate induction time scale

Not ideal for end productRequires microscopyHowever, excitation wavelength is ~520 nm, so UV exposure is not an issue


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The Erasing Circuit1. Reporters

2. The ON Switch

3. Heat-inducible reporters: 1 + 2

Test Circuits (I)Test Circuits (I)The Writing Circuit1. Reporters

2. The OFF Switch

3. UV-inducible reporters: 1 + 2


mCherrymCherry434434OOPPRMRM

venusvenus434434OOPPRMRM

434434lacOlacOPPRMRM

venusvenuscIcIOOPPRMRM

venusvenuslacOlacOPPRMRM

lacIlacItsts434434OOlacPlacP


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Test Circuits (II)Test Circuits (II)All Together Now:

434-CI857 Switch

434-LacIts Switch




434434lacOlacOPPRMRM lacIlacItsts434434OOlacPlacP


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Timing Diagram: The 434-Timing Diagram: The 434-CI587 SwitchCI587 SwitchWriting (?)

1. UV irradiation (1-10s)

2. RecA activation (10min-2h)

3. 434 degradation (?)4. Switching ON:

CI587 induction (?)mCherry induction (?)1. Transcription (?)2. Translation (?)3. Folding (~15min)

Erasing (?)1. Heat application (?)2. CI587 denaturing (?)3. Switching OFF:

434 induction (?)1. Transcription (?)2. Translation (?)3. Folding (?)

mCherry degradation (?)


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Parts to GetParts to GetOFF Switch:

434: BBa_C0052434-repressed PRM promoter: BBa_I12036

ON Switch:cI857: BBa_C0081CI-repressed PRM promoter: BBa_R0051

lacIts: (?)LacI-repressed promoter: BBa_R0011

Ink:mCherry: BBa_E2060 (yeast)venus: BBa_E2030 (yeast)

Strain:lacI –: Bba_V1002

http://parts.mit.edu/ [2005-06-12]http://parts.mit.edu/ [2005-06-12]


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What We Don't KnowWhat We Don't KnowThe SOS response requires DNA replication; does that mean the cells have to be in exponential growth phase?Which parts should we use?

Promoters and operators.RBSs and terminators.Vectors.Strains: If we use LacI(ts), the strain must be lacI –. What are the strain requirements of the other circuits?

What are the kinetics of ON switch/OFF switch inductions? Reporters?How do we go about modeling these things?

What do we have to know before we can model? What do we have to model before we know what to test?


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AcknowledgmentsAcknowledgmentsPam SilverGeorge ChurchKit ParkerRadhika NagpalAlain Viel

Ira PhilipsSasha Wait

Orr AshenbergPatrick BradleyConnie ChengKang-Xing JinPaula NunesDanny PopperSasha Rush

BioSketch The bacterial sketch pad.

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