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2/45 1. Tri-Stable Switch 2. Lead Detector 3. iGEM Community
Tri-Stable Switch Adam Emrich, Norris Hung, Kyle Schutter Inspiration Architecture Modeling Applying the Model Status
Inspiration
Gardner, Cantor, & Collins in Nature 2000 Our Role Model
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
ModelProduct
Architecture
Black Box aTc IPTG Arabinose GFP RFP YFP
P AraC/BAD TetR Lac I TetR AraC P Lac I P Tet R GFP YFP RFP L-arabinose IPTG aTc
Modeling
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
Applying the model
β=1 β=10 β=2 β=3 Rate of Change of Repressor vs Repressor Concentration for Different Cooperativity Levels dY (X or Z)
Experimentally Determine Model Parameters
How Can the Tri-Stable Switch be used? C A B GFP YFP RFP Protein 1 Protein 2 Protein 3
Applications Basic Circuit Component Memory (Trinary?) Drug Delivery Cell Differentiation
α=? Summer Goals Design Genetic Architecture Derive Model Experimentally Determine Model Parameters Test and Build Switch Summer Accomplishments tests designed ligations underway
Lead Detector Deepa Galaiya, Jeff Hofmann, Rohan Maddamsetti Inspiration Architecture Part Characterization Progress
Inspiration
Contaminated Water: __% Clean Water: __% Contaminated Water: 40% Clean Water: 60% WHO Inspiration
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
24/45 Architecture
25/45 Simple Lead Detector Fluorescent Protein Lead Promoter Lead Binding Protein
26/45 Low Fluorescence
27/45 Solution: Amplify the Signal Fluorescent Protein Lead Promoter Amplifier Lead Binding Protein
28/45 Amplified Fluorescence Low Fluorescence
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
30/45 Part Characterization
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:
32/45 AHL = Signal Lead Promoter Amplifier LuxI AHL Output AHL Input Match Output to Input to complete circuit. PbrR AHL Assay
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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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
36/45 Hypothesis: Direct relationships between AHL and GFP Result: Inverse relationship between AHL and GFP 3. Characterize Amplifier
37/45 Inverse relationship between AHL input and GFP output
Amplifier Doesn’t Work! Why? Hypothesis: AHL molecules affect Fluorescence
39/45 Simple Lead Detector Fluorescent Protein Lead Promoter Lead Binding Protein
40/45 Develop AHL Assay Characterize Amplifier Characterize New Lead Parts Summer Accomplishments
Community
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43/45 Tri-Stable Switch Applying a Model Lead Detector Part Characterization Community Highschoolers Synthetic Biology class Brown iGEM
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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