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BioSketch The bacterial sketch pad.

BioSketch The bacterial sketch pad. Chris Doucette, Thomas Noriega, Hing Eng, Yves Wang, Jennifer Gao, Yin Li Group Meeting 2005-06-13. The Idea. Write with UV light. Erase by turning up the heat. Remember with a genetic switch. Play with bacteria! Advance synthetic biology.

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BioSketch The bacterial sketch pad.

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  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! OFF Switch ON Switch

  4. OFF to ON: Writing OFF to ON: Writing Pen Ink OFF Switch ON Switch

  5. OFF to ON: Alternative Schemes Pen Ink OFF Switch ON Switch

  6. ON to OFF: Erasing ON to OFF: Erasing Ink OFF Switch ON Switch Eraser

  7. ON to OFF: Alternative Schemes Ink OFF Switch ON 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. EMBOJ 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. EMBOJ 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.

  12. UV ssDNA ON ONP 434O oNswitch RecA* PRM ONO 434 Turning On the Switch with UV dsDNA RecA 434 Kobayashi et al. 2004. PNAS 101: 8414-8419; Yasuda et al., 1998. EMBOJ 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

  14. 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: 6568–6577.) • Tet (Wissmann et al. 1991. Genetics 128: 225-232.) • AraC (Irr & Englesberg. 1971. J Bacteriol 105: 136-141.) • Thermo-labile mRNAs

  15. 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.

  16. 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.

  17. PRM 434O l cI857 PRM cIO 434 Turning Off the Switch with Heat 434 CI857 CI857-

  18. 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

  19. 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.

  20. 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

  21. PRM cIO venus PRM lacO venus PRM cIO 434 PRM lacO 434 PRM 434O venus PRM 434O l cI857 lacP 434O lacIts PRM 434O mCherry Test Circuits (I) • The Writing Circuit • Reporters • The OFF Switch • UV-inducible reporters: 1 + 2 • The Erasing Circuit • Reporters • The ON Switch • Heat-inducible reporters: 1 + 2

  22. PRM cIO 434 PRM lacO 434 PRM 434O l cI857 lacP 434O lacIts PRM PRM 434O 434O mCherry mCherry Test Circuits (II) • All Together Now: • 434-CI857 Switch • 434-LacIts Switch

  23. 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 (?)

  24. 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]

  25. 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?

  26. 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

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