Post on 25-Jan-2016
description
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
22
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?
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
33
The Toggle Switch: It The Toggle Switch: It Remembers!Remembers!
ON SwitchON SwitchOFF SwitchOFF Switch
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
44
PenPen
OFF OFF to ON: Writingto ON: Writing
ON SwitchON Switch InkInkOFF SwitchOFF Switch
OFF to OFF to ONON: Writing: Writing
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
55
OFF to ON: Alternative OFF to ON: Alternative SchemesSchemes
ON SwitchON Switch InkInk
PenPen
OFF SwitchOFF Switch
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
66
ONON to OFF: Erasing to OFF: Erasing
ON SwitchON Switch InkInk
EraserEraser
OFF SwitchOFF Switch
ONON to to OFFOFF: Erasing: Erasing
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
77
ON to OFF: Alternative ON to OFF: Alternative SchemesSchemes
ON SwitchON Switch InkInk
EraserEraser
OFF SwitchOFF Switch
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
88
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.
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
99
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.
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
1010
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.
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
1111
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.
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
1212
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
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
1313
The EraserThe EraserProperties
Effects reversibleEasily controlledLow spatial resolution
CandidatesChemical signalsTemperature/heat
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
1414
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
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
1515
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.
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
1616
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.
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
1717
Turning Off the Switch with Turning Off the Switch with HeatHeat
CI857CI857CI857CI857--
434434
434434cIcIOOPPRMRM cI857cI857434434OOPPRMRM
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
1818
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
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
1919
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.
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
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
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
2121
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
434434cIcIOOPPRMRM cI857cI857434434OOPPRMRM
mCherrymCherry434434OOPPRMRM
venusvenus434434OOPPRMRM
434434lacOlacOPPRMRM
venusvenuscIcIOOPPRMRM
venusvenuslacOlacOPPRMRM
lacIlacItsts434434OOlacPlacP
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
2222
Test Circuits (II)Test Circuits (II)All Together Now:
434-CI857 Switch
434-LacIts Switch
434434cIcIOOPPRMRM cI857cI857434434OOPPRMRM
mCherrymCherry434434OOPPRMRM
mCherrymCherry434434OOPPRMRM
434434lacOlacOPPRMRM lacIlacItsts434434OOlacPlacP
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
2323
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 (?)
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
2424
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]
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
2525
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?
BioSketchBioSketch Harvard iGEM 2005Harvard iGEM 2005
2626
AcknowledgmentsAcknowledgmentsPam SilverGeorge ChurchKit ParkerRadhika NagpalAlain Viel
Ira PhilipsSasha Wait
Orr AshenbergPatrick BradleyConnie ChengKang-Xing JinPaula NunesDanny PopperSasha Rush