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Published byMelinda Mason Modified over 9 years ago
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2/45 1. Tri-Stable Switch 2. Lead Detector 3. iGEM Community
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Tri-Stable Switch Adam Emrich, Norris Hung, Kyle Schutter Inspiration Architecture Modeling Applying the Model Status
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Inspiration
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Gardner, Cantor, & Collins in Nature 2000 Our Role Model
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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
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ModelProduct
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Architecture
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Black Box aTc IPTG Arabinose GFP RFP YFP
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P AraC/BAD TetR Lac I TetR AraC P Lac I P Tet R GFP YFP RFP L-arabinose IPTG aTc
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Modeling
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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
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Applying the model
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β=1 β=10 β=2 β=3 Rate of Change of Repressor vs Repressor Concentration for Different Cooperativity Levels dY (X or Z)
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Experimentally Determine Model Parameters
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How Can the Tri-Stable Switch be used? C A B GFP YFP RFP Protein 1 Protein 2 Protein 3
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Applications Basic Circuit Component Memory (Trinary?) Drug Delivery Cell Differentiation
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α=? Summer Goals Design Genetic Architecture Derive Model Experimentally Determine Model Parameters Test and Build Switch Summer Accomplishments tests designed ligations underway
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Lead Detector Deepa Galaiya, Jeff Hofmann, Rohan Maddamsetti Inspiration Architecture Part Characterization Progress
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Inspiration
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Contaminated Water: __% Clean Water: __% Contaminated Water: 40% Clean Water: 60% WHO Inspiration
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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
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24/45 Architecture
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25/45 Simple Lead Detector Fluorescent Protein Lead Promoter Lead Binding Protein
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26/45 Low Fluorescence
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27/45 Solution: Amplify the Signal Fluorescent Protein Lead Promoter Amplifier Lead Binding Protein
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28/45 Amplified Fluorescence Low Fluorescence
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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
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30/45 Part Characterization
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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:
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32/45 AHL = Signal Lead Promoter Amplifier LuxI AHL Output AHL Input Match Output to Input to complete circuit. PbrR691 1. AHL Assay
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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
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36/45 Hypothesis: Direct relationships between AHL and GFP Result: Inverse relationship between AHL and GFP 3. Characterize Amplifier
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37/45 Inverse relationship between AHL input and GFP output
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Amplifier Doesn’t Work! Why? Hypothesis: AHL molecules affect Fluorescence
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39/45 Simple Lead Detector Fluorescent Protein Lead Promoter Lead Binding Protein
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40/45 Develop AHL Assay Characterize Amplifier Characterize New Lead Parts Summer Accomplishments
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Community
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43/45 Tri-Stable Switch Applying a Model Lead Detector Part Characterization Community Highschoolers Synthetic Biology class Brown iGEM
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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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