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2. 2/45 1. Tri-Stable Switch 2. Lead Detector 3. iGEM Community

3. Tri-Stable Switch Adam Emrich, Norris Hung, Kyle Schutter Inspiration 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. ModelProduct

8. Architecture

9. Black Box aTc IPTG Arabinose GFP RFP YFP

10. P AraC/BAD TetR Lac I TetR AraC P Lac I P Tet R GFP YFP RFP L-arabinose IPTG aTc

11. Modeling

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

13. Applying the model

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

16. Experimentally Determine Model Parameters

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

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

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

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

21. Inspiration

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

23. 23/45 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. 24/45 Architecture

25. 25/45 Simple Lead Detector Fluorescent Protein Lead Promoter Lead Binding Protein

26. 26/45 Low Fluorescence

27. 27/45 Solution: Amplify the Signal Fluorescent Protein Lead Promoter Amplifier Lead Binding Protein

28. 28/45 Amplified Fluorescence Low Fluorescence

29. 29/45 Architecture pTet (Constitutive On) PbrR691 Lead Promoter PbrR691 LuxI GFP LuxR AHL = Cell Signaling Molecule Made by LuxI To other cells AHL Binding Protein/Promoter Complex + Amplifier AHL Producer Fluorescent

30. 30/45 Part Characterization

31. 31/45 Lead Promoter Amplifier 1.Develop Assay to Measure AHL 2.Characterize New Parts: Lead Promoter and Lead Binding Protein 3.Characterize Amplifier 3 2 Experimental Steps:

32. 32/45 AHL = Signal Lead Promoter Amplifier LuxI AHL Output AHL Input Match Output to Input to complete circuit. PbrR691 1. AHL Assay

33. 33/45 How did we measure the AHL Signal? AHL to GFP Converter (Part T9002) 1. AHL Assay AHLGFP AHL to GFP Converter

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35. 2. New Lead Parts Lead Promoter Lead Binding Protein 4 New Parts: 1.Lead Promoter 2.Lead Binding Protein 3.2 Lead Binding Proteins under native promoter

36. 36/45 Hypothesis: Direct relationships between AHL and GFP Result: Inverse relationship between AHL and GFP 3. Characterize Amplifier

37. 37/45 Inverse relationship between AHL input and GFP output

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

39. 39/45 Simple Lead Detector Fluorescent Protein Lead Promoter Lead Binding Protein

40. 40/45 Develop AHL Assay Characterize Amplifier Characterize New Lead Parts Summer Accomplishments

41. Community

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43. 43/45 Tri-Stable Switch Applying a Model Lead Detector Part Characterization Community Highschoolers Synthetic Biology class Brown iGEM

44. 44/45 John Cumbers Alex Brodsky Tayhas Palmore Gary Wessel Multidisciplinary Lab CCMB MCB MPPB Department of Physics Division of Engineering

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