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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. General Design. Lead. Lead Detection.
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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
General Design Lead Lead Detection Lead Detection Signal Amplification Signal Amplification Fluorescent Output 4/55
Lead Sensor Lead Detection 6/55
In Ralstonia metallidurans: PbrR691 Coding Region Non-Coding Region PbrA Coding Region 7/55
PbrR691 Promoter PbrR691 Coding Region RBS Non-Coding Region RBS PbrA Coding Region Lead Promoter 8/55
1 version 15 total 3 versions PbrR691 Coding Region Non-Coding Region PbrA Coding Region 3 versions 6 versions (Neils method) 2 versions 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:Indirectrelationship 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
Input C Input A Input B Output C Output B Output A Achieving Tri-stability State A State B State C 26/55
The Switch The Architecture as planned A pBAD LacI TetR B pLac AraC TetR C pTet LacI AraC 27/55
The Switch The Architecture as planned L-arabinose pBAD LacI TetR pLac AraC TetR pTet LacI AraC 28/55
The Switch The Architecture as planned pBAD LacI TetR IPTG pLac AraC TetR pTet LacI AraC 29/55
The Switch The Architecture as planned pBAD LacI TetR pLac AraC TetR anhydrotetracycline pTet LacI AraC 30/55
AraC pC pBAD TetR LacI pLac AraC TetR pTet LacI AraC Architecture ReDesigned • Parts in the registry only allow two stable states and a third inducible state • pBAD promoter is attached to gene 31/55
Architecture ReDesigned L-arabinose AraC pC pBAD TetR LacI pLac AraC TetR pTet LacI AraC 32/55
Architecture ReDesigned L-arabinose AraC pC pBAD TetR LacI pLac AraC TetR pTet LacI AraC But there is a lot of araC in the system 33/55
Architecture ReDesigned AraC pC pBAD TetR LacI pLac AraC TetR pTet LacI AraC 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
Transformation of DNA, to make more DNA 3 Major Steps to Create Parts 38/55
Transformation Restriction Ligation The 3 Major Steps 39/55
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. Purposes of each Step 40/55
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. Step 1: Transformation 41/55
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. Step 2: Restriction 42/55
Purpose: To attach DNA from Restriction step together, resulting in new Genetic Parts. Method: ??? We do not have an exact method for this yet. Step 3: Ligation 43/55
Transformation: ~Early July Competent Cell Preparation: ~Early August Ligation: ? Protocol Determination 44/55
Our ligation success rate is about 10%. We are currently running experiments to determine a protocol that works at a higher success rate. Step 3: Ligation Protocol 45/55
1. Establish a working protocol for Ligations 2. Work on project during semester 3. Assemble a Bi-Stable Switch by the Jamboree Plan for the Future 46/55
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