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Harvard iGEM 2005 - BioSketch Team

BioSketch: A bacterial sketchpad driven by a UV- and heat-sensitive genetic toggle switch. Harvard iGEM 2005 - BioSketch Team Christopher Doucette, Hing Eng, Jennifer Gao, Yin Li, Thomas Noriega, Yves Wang;

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Harvard iGEM 2005 - BioSketch Team

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  1. BioSketch:A bacterial sketchpad drivenby a UV- and heat-sensitive genetic toggle switch Harvard iGEM 2005 - BioSketch Team Christopher Doucette, Hing Eng, Jennifer Gao, Yin Li, Thomas Noriega, Yves Wang; George Church, Radhika Nagpal, Kevin Parker, Ira Phillips, Pamela Silver, Alain Viel, Alexander Wait

  2. BACTERIA HEAT UV Goal • Turn a lawn of E. coli into a reusable sketch pad • Write with a UV pen • Erase with heat

  3. 40 °C 30 °C Project Concept

  4. UV A B REPORTER HEAT • Writing: A is cleaved in response to UV, allowing expression of B and reporter • Erasing: Heat inactivates B, allowing expression of A Circuit Design: Toggle Switch • A and B are transcriptional repressors

  5. UV Reporter Plasmid PL* gfp l cI Ptrc PL* lacI Toggle Switch Plasmid IPTG Kobayashi, Kaern et al.”Programmable Cells: Interfacing natural and engineered gene networks.” PNAS 101(22): 8414–8419 2004. The Toggle Switch Plasmid Original design from Collins lab:

  6. The Toggle Switch Plasmid Modified design for BioSketch: UV Reporter Plasmid PL* gfp l cI Ptrc PL* lacIts Toggle Switch Plasmid IPTG, Heat Kobayashi, Kaern et al.”Programmable Cells: Interfacing natural and engineered gene networks.” PNAS 101(22): 8414–84192004.

  7. Collins Switch PL* lacI 40 °C Switch 37 °C Switch PL* PL* lacIts 241 lacIts 265 Adding Heat Sensitivity • Two temperature sensitive mutants of LacI repressor • Ala 241  Thr: Repression lifted at 40 °C • Gly 265  Asp: Repression lifted at 37 °C • Site-directed mutagenesis of Collins plasmid

  8. Testing the Circuit • Two l cI / lacIts switches built • Can we set the OFF state with heat? • Can we set the ON state with UV? • Are states maintained over time? • Assays: • Fluorescence microscopy • FACS analysis

  9. Experimental Design Day 1 30°C o.n. Day 2 30, 32, 35, 37, or 40°C o.n. Day 3 Assay Testing: Turning Switch OFF • Strains (LacI-) • 40 °C or 37 °C Switch • ON state Control: GFP reporter only • OFF state Control: No construct

  10. 40 °C Switch 30°C  30°C 30°C  32°C 30°C  37°C 30°C  40°C GFP only No construct • Incubation at 37-40°C almost completely abolishes fluorescence. • Increasing temperature had no effect on fluorescence profiles of control strains. Testing: Turning Switch OFF

  11. Experimental Design Day 1 40°C o.n. Day 2 0, 6, 12, 24, 48 J/m2 4h @ 30°C Assay Testing: Turning Switch ON • Strains (LacI-) • 40 °C or 37 °C Switch • Positive Control: GFP reporter only • Negative Control: No construct

  12. 40 °C Switch 0 J/m2 6 J/m2 24 J/m2 48 J/m2 • Increasing intensity of UV correlates with increasing intensity of cell fluorescence. Testing: Turning Switch ON • UV irradiation had no effect on fluorescence profiles of control strains.

  13. Testing: State Maintenance • 3 states for cell populations • ON (after UV exposure) • OFF (after heat exposure) • Mixed (some ON, some OFF) • Populations in mixed state initially • After cells are set to ON or OFF, state should be stable indefinitely

  14. 40 °C Switch No heat treatment 2 hrs after heat 24 hrs after heat • Population begins in mixed state. • 2 hours after incubation, population is set to OFF state. • 24 hours after incubation, population returns to mixed state. Testing: State Maintenance

  15. How To Improve State Maintenance • For bistability, rates of repression and expression must be carefully balanced • Promoter strength & leakiness • Ribosome binding site strength • Need to test multiple combinations of various promoters and RBSs • Send in the BioBricks!

  16. l cI / lacIts switch: PL lacIts Plac l cI PL mCherry • 434 cI / l cIts switch: P434 l cIts PL 434 cI P434 mCherry New Plasmid Designs • Build from scratch using BioBricks from MIT Registry

  17. Summary • Accomplished: • Turn switch ON with UV • Turn switch OFF with heat • To Do List: • Get better induction with UV • More robust state-maintenance • Vary RBS, promoter strength • Test on solid media

  18. Acknowledgments • Orr Ashenberg, Patrick Bradley, Connie Cheng, Kang-Xing Jin, Danny Popper, Sasha Rush • Alain Viel, Ira Phillips, Sasha Wait • Pam Silver, George Church, Kit Parker, Radhika Nagpal • Harvard Provost Office, HHMI, DEAS, Division of Life Sciences • Jim Collins Lab (Boston University) • Registry of Standard Biological Parts (MIT) • Bauer Center Flow Cytometry Facility (Harvard) • Dana Farber Cancer Institute Flow Cytometry Facility

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