1. Tri-Stable Switch 2. Lead Detector 3. iGEM Community

2. 1. Tri-Stable Switch 2. Lead Detector 3. iGEM Community

3. Inspiration Tri-Stable SwitchAdam Emrich, Norris Hung, Kyle Schutter Architecture Modeling Applying the Model Status

4. Inspiration

5. Gardner, Cantor, & Collins in Nature 2000 Our Role Model

6. n-Stable Switch “If I have seen a little further it is by standing on the shoulders of Giants." - Isaac Newton • Apply their logic • - Apply their model • - Use iGem Parts

7. Model Product

8. Architecture

9. GFP Arabinose Black Box IPTG RFP aTc YFP

10. L-arabinose IPTG aTc PAraC/BAD TetR LacI GFP PLacI AraC TetR RFP PTetR LacI AraC YFP

11. Modeling

12. β = cooperativity of repression α = repressor production rate x = repressor1 y = repressor2 z = repressor3 Less Repression by Other Repressors Degradation Change in Repressor Repressor Production The Model

13. Applying the model

14. Rate of Change of Repressor vs Repressor Concentration for Different Cooperativity Levels dY β=1 β=2 β=3 β=10 (X or Z)

16. Experimentally Determine Model Parameters

17. How Can the Tri-Stable Switch be used? A GFP Protein 1 B RFP Protein 2 C YFP Protein 3

18. Applications Basic Circuit Component Memory (Trinary?) Drug Delivery Cell Differentiation

19. Summer Accomplishments Summer Goals Design Genetic Architecture Derive Model Experimentally Determine Model Parameters Test and Build Switch α=? tests designed ligations underway

20. Inspiration Architecture Lead DetectorDeepa Galaiya, Jeff Hofmann, Rohan Maddamsetti Part Characterization Progress

21. Inspiration

22. Inspiration Contaminated Water: __% Clean Water: __% Contaminated Water: 40% Clean Water: 60% WHO

23. Chen P, Greenberg B, Taghavi S, Romano C, van der Lelie D, He C (2005) An exceptionally selective lead(II)-regulatory protein from Ralstonia metallidurans: development of a fluorescent lead(II) probe. Angew Chem Int Ed Engl 44:2715–2719

24. Architecture

25. Simple Lead Detector Lead Binding Protein Fluorescent Protein Lead Promoter

26. Low Fluorescence

27. Solution: Amplify the Signal Lead Binding Protein Fluorescent Protein Amplifier Lead Promoter

28. Low Fluorescence Amplified Fluorescence

29. Architecture To other cells To other cells AHL = Cell Signaling Molecule Made by LuxI LuxR PbrR691 PbrR691 LuxR pTet (Constitutive On) + LuxI LuxI GFP LuxR Amplifier AHL Binding Protein/Promoter Complex Lead Promoter AHL Producer AHL Producer Fluorescent

30. Part Characterization

31. Experimental Steps: • Develop Assay to Measure AHL • Characterize New Parts: Lead Promoter and Lead Binding Protein • Characterize Amplifier 2 3 Amplifier Lead Promoter

32. 1. AHL Assay AHL = Signal LuxI PbrR691 Amplifier Lead Promoter AHL Output AHL Input Match Output to Input to complete circuit.

33. 1. AHL Assay How did we measure the AHL Signal? AHL to GFP Converter (Part T9002) AHL AHL to GFP Converter GFP

35. 2. New Lead Parts Lead Binding Protein Lead Promoter 4 New Parts: Lead Promoter Lead Binding Protein 2 Lead Binding Proteins under native promoter

36. 3. Characterize Amplifier Hypothesis: Direct relationships between AHL and GFP Result: Inverse relationship between AHL and GFP

37. Inverse relationship between AHL input and GFP output

38. Amplifier Doesn’t Work! Why? Hypothesis: AHL molecules affect Fluorescence

39. Simple Lead Detector Lead Binding Protein Fluorescent Protein Lead Promoter

40. Summer Accomplishments Develop AHL Assay Characterize Amplifier Characterize New Lead Parts

41. Community

43. Brown iGEM Tri-Stable Switch Applying a Model Lead Detector Part Characterization Community Highschoolers Synthetic Biology class

44. John CumbersAlex BrodskyTayhas PalmoreGary WesselMultidisciplinary LabCCMBMCBMPPB Department of Physics Division of Engineering

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