Brown iGEM international genetically engineered machines competition August Update 1/55

Brown iGEM 2007 Lead Sensor Tristable Switch iGEM Jamboree on November 4th at MIT 2/55

Lead Sensor Introduction Speaker: Deepa Galaiya 3/55

Lead Detection Signal Amplification Fluorescent Output Lead General Design Lead Detection Signal Amplification 4/55

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Lead Sensor Lead Detection 6/55

PbrR691 Coding Region PbrA Coding Region Non-Coding Region In Ralstonia metallidurans: 7/55

PbrR691 Coding Region PbrA Coding Region Non-Coding Region RBS Lead Promoter PbrR691 Promoter 8/55

PbrR691 Coding Region PbrA Coding Region Non-Coding Region 3 versions 6 versions (Neils method) 3 versions 1 version 2 versions 15 total 9/55

Planned Ligations -All 15 parts into BioBrick plasmid -pTet to PbrR691 alone -All promoters and PbrR691 combinations to LuxI -All promoters and PbrR691 combinations to GFP -pTet-PbrR691 to promoters-LuxI -pTet-PbrR691 to promoters-GFP 10/55

Completed Ligations -All 15 parts into BioBrick plasmid -pTet to PbrR691 alone (done today!) -All promoters and PbrR691 combinations to LuxI -All promoters and PbrR691 combinations to GFP -pTet-PbrR691 to promoters-LuxI -pTet-PbrR691 to promoters-GFP 11/55

Results 15 parts into Biobrick gelled and sequenced PbrR691/promoter combinations in presence of lead nitrate give no GFP production and no AHL production compared to control. 12/55

Discussion & Future Plans PbrR691 most likely not expressed in combination parts. (Transcription factors needed?). Promoter not abandoned yet! Constitutive expression under pTet possible. Neils’ group was able to overexpress protein in E Coli under IPTG control. 13/55

Lead Sensor The Amplifier Speaker: Jeff Hofmann 14/55

T9002 J37015 (The Amplifier) Differences:  Positive Feedback Loop  Stronger Ribosome Binding Site 15/55

How did we measure this? GFP fluorescence – average control GFP fluorescence Cell Density 16/55

Expected: Amplifier produces more GFP than T9002 Results: T9002 produces far more GFP than Amplifier (GFP – Control) / Cell Density Time (hours) 17/55

Expected: Direct relationship between AHL input and GFP output Results: Indirect relationship between AHL input and GFP output 18/55

More AHL = Less GFP! 19/55

Why does this happen?  Possible wrong promoter  GFP is further away from promoter in J37015 T9002 J37015 (The Amplifier) 20/55

Why does this happen?  Possible wrong promoter  GFP is further away from promoter in J37015 T9002 J37015 (The Amplifier) 21/55

Lead Sensor Sequencing Speaker: Rohan Maddamsetti 22/55

Sequencing Importance of Sequencing Sequencing the Amplifier Sequencing the Lead Parts Where do we go from here? 23/55

Tri-Stable Switch Speaker: Kyle Schutter 24/55

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Achieving Tri-stability State A State C State B Output A Output B Output C Input A Input C Input B 26/55

The Switch pBAD TetR LacI TetR AraC pLac pTet The Architecture as planned C A B 27/55

The Switch pBAD TetR LacI TetR AraC pLac pTet The Architecture as planned L-arabinose 28/55

The Switch pBAD TetR LacI TetR AraC pLac pTet The Architecture as planned IPTG 29/55

The Switch pBAD TetR LacI TetR AraC pLac pTet The Architecture as planned anhydrotetracycline 30/55

Parts in the registry only allow two stable states and a third inducible state pBAD promoter is attached to gene pBAD TetR LacI TetR AraC pLac pTet pC AraC Architecture ReDesigned 31/55

pBAD TetR LacI TetR AraC pLac pTet pC AraC Architecture ReDesigned L-arabinose 32/55

pBAD TetR LacI TetR AraC pLac pTet pC AraC Architecture ReDesigned L-arabinose But there is a lot of araC in the system 33/55

pBAD TetR LacI TetR AraC pLac pTet pC AraC Architecture ReDesigned The system will fall into whichever of the other two states is stronger. 34/55

Characterization Registry not as well characterized/reliable as expected – Rumor that araC gene has promoter region inside it – All repressors LVA tagged: fast degradation leads to poor repression – AraC connected to pBAD promoter 35/55

iGEM Characterization Protocol Starting to formulate Characterization protocol for Promoters and Repressors – Promoters: relative, how “on” or “off” – Repressors: cooperativity, binding constant 36/55

Tristable Switch Creating the Parts Speaker: Adam Emrich 37/55

3 Major Steps to Create Parts Transformation of DNA, to make more DNA 38/55

The 3 Major Steps Transformation Restriction Ligation 39/55

Purposes of each Step Transformation: Increases the amount of DNA. Extraction: Removes DNA from cells. Restriction: Cuts DNA, to prepare it for Ligation. Ligation: Attaches cut DNA, to create new genetic parts. 40/55

Step 1: Transformation Purpose: To create more DNA. Method: 1. Insert DNA into specially prepared Competent Cells. 2. Plate out cells, allow to grow overnight. 3. Create overnight culture. 4. Extract DNA. 41/55

Step 2: Restriction Purpose: To cut DNA from Step 1, in preparation for Step 3: Ligation. Method: 1. Insert DNA into a vial. 2. Add buffer and restriction enzymes, incubate 4-6 hours. 3. Heat inactivate enzymes. 42/55

Step 3: Ligation Purpose: To attach DNA from Restriction step together, resulting in new Genetic Parts. Method: ??? We do not have an exact method for this yet. 43/55

Protocol Determination Transformation: ~Early July Competent Cell Preparation: ~Early August Ligation: ? 44/55

Step 3: Ligation Protocol Our ligation success rate is about 10%. We are currently running experiments to determine a protocol that works at a higher success rate. 45/55

Plan for the Future 1. Establish a working protocol for Ligations 2. Work on project during semester 3. Assemble a Bi-Stable Switch by the Jamboree 46/55

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The Next Step Preparing for November 4th, MIT Continue progress Build Characterize 48/55

Next Year Master protocols earlier Transformation to Ligation Problem-solving skills 49/55

New Technologies Automated Assembly Synthesize all DNA New Equipment - Plate Reader 50/55

Student Led iGEM Student Group (Fall ‘07) 2008 Applications judged by 2007 Team Be Selective Goal: 50 Applications 51/55

Sponsors Thank you 52/55

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John Cumbers Alex Brodsky Tayhas Palmore Gary Wessel Multidisciplinary Lab (Kathy, Shirley, Faye, Phil, Sarah) CCMB MCB MPPB Department of Physics Division of Engineering 54/55

Brown iGEM international genetically engineered machines competition August Update 55/55