BioSketch The bacterial sketch pad.

1
BioSketchThe bacterial sketch pad.

• Chris Doucette, Thomas Noriega, Hing Eng,
• Yves Wang, Jennifer Gao, Yin Li
• Group Meeting
• 2005-06-13

2
The Idea

• Write 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?

3
The Toggle Switch It Remembers!
ON Switch
OFF Switch
4
OFF to ON Writing
OFF to ON Writing
Pen
Ink
ON Switch
OFF Switch
5
OFF to ON Alternative Schemes
Pen
Ink
ON Switch
OFF Switch
6
ON to OFF Erasing
ON to OFF Erasing
Ink
ON Switch
OFF Switch
Eraser
7
ON to OFF Alternative Schemes
Ink
ON Switch
OFF Switch
Eraser
8
The Pen

• Required Properties
• Rapidly induces response/short exposure
• Transient/reversible
• High spatial resolution
• Candidates
• Chemical signals
• AHL
• IPTG
• Electromagnetic waves
• Infrared (heat)
• UV radiation (254nm)

• Devices
• Chemical signals
• Microspray
• Electromagnetic waves
• Laser
• Microscope
• Lamp stencil

Kobayashi et al. 2004. PNAS 101 8414-8419.
9
The OFF Switch

• Required Properties
• Rapidly repressible by Pen
• OR
• Inducible by Eraser
• Antagonistic to ON Switch
• Candidates
• 434 repressor
• l CI repressor
• LexA 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. EMBO J 17 3207-3216
Gardner et al., 2000. Nature 403 339-342
Salles Paoletti. 1983. PNAS 80 65-69.
10
The l CI Repressor

• Properties
• 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. EMBO J 17 3207-3216
Gardner et al., 2000. Nature 403 339-342.
11
The 434 repressor

• Properties
• 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. Nucleic Acids Res 27
3474-3480.
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Turning On the Switch with UV
dsDNA
RecA
434
Kobayashi et al. 2004. PNAS 101 8414-8419
Yasuda et al., 1998. EMBO J 17
3207-3216 Higashitani et al. 1995. J Bacteriol
177 3610-3612 Salles Paoletti. 1983. PNAS 80
65-69 Pawlowski Koudelka. 2004. J Bacteriol
186 1-7.
13
The Eraser

• Properties
• Effects reversible
• Easily controlled
• Low spatial resolution
• Candidates
• Chemical signals
• Temperature/heat

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The ON Switch

• Required Properties
• Repressible by Eraser
• OR
• Rapidly inducible by Pen
• Antagonistic to ON Switch component
• Candidates
• Temperature-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.)
• l 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
  65686577.)
• 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) Repressor

• Properties
• Loses capacity to repress at 42? C
• Represses promoters containing lacO
• WT lacI has been used in genetic toggle switches,
  and other synthetic circuits.
• Several Variants
• Ser300?Asn (Yabuta et al., 1995)
• Gly187?Ser (Chao et al., 2002)
• Leu233?Lys (Chao et al., 2002)
• Ala241?Thr (Chao et al., 2002 Yabuta et al.,
  1995)
• Gly265?Asp (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. 1995. J Biotechnol 39 67-73 Chao et al.
2002. Biotechnol Bioeng 79 1-8 Bukrinsky et al.
1988. Gene 70 415-417.
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The l CI857 Repressor

• Properties
• 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 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 Heat
434
CI857
CI857-
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The Ink

• Properties
• Repressed by OFF switch
• OR
• Induced by ON switch
• Rapidly inducible (preferred)
• Rapidly degraded (preferred)
• Reversible
• Candidates
• mCherry monomeric dsRed derivative, rapid
  folding, shows up purple in visible light
• Venus rapidly-folding (2 min) YFP variant
• Ca2-sensitive GFP
• cAMP-sensitive GFP

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mCherry

• Rapid folding
• Monomeric, as opposed to tetrameric dsRed
• Developed by directed evolution - selection for
  maturation speed and brightness
• 50 of maximum fluorescence 15 min.
  post-translation
• Excited by visible light

• Visible without microscopy
• Advantageous - UV excitation could interfere with
  drawing
• More immediate results for user

Shaner et al. 2004 Nature Biotechnol 22
1567-1572.
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Venus YFP

• Extremely 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 product
• Requires microscopy
• However, excitation wavelength is 520 nm, so UV
  exposure is not an issue

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Test Circuits (I)

• The Erasing Circuit
• Reporters
• The ON Switch
• Heat-inducible reporters 1 2

• The Writing Circuit
• Reporters
• The OFF Switch
• UV-inducible reporters 1 2

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Test Circuits (II)

• All Together Now
• 434-CI857 Switch
• 434-LacIts Switch

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Timing Diagram The 434-CI587 Switch

• Writing (?)
• UV irradiation (1-10s)
• RecA activation (10min-2h)
• 434 degradation (?)
• Switching ON
• CI587 induction (?)
• mCherry induction (?)
• Transcription (?)
• Translation (?)
• Folding (15min)

• Erasing (?)
• Heat application (?)
• CI587 denaturing (?)
• Switching OFF
• 434 induction (?)
• Transcription (?)
• Translation (?)
• Folding (?)
• mCherry degradation (?)

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Parts to Get

• OFF Switch
• 434 BBa_C0052
• 434-repressed PRM promoter BBa_I12036
• ON Switch
• lcI857 BBa_C0081
• l CI-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
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What We Don't Know

• The 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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Acknowledgments

• Pam Silver
• George Church
• Kit Parker
• Radhika Nagpal
• Alain Viel
• Ira Philips
• Sasha Wait

• Orr Ashenberg
• Patrick Bradley
• Connie Cheng
• Kang-Xing Jin
• Paula Nunes
• Danny Popper
• Sasha Rush