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Brown iGEM

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Brown iGEM

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

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

  3. Lead Sensor Introduction Speaker: Deepa Galaiya 3/55

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

  5. 5/55

  6. Lead Sensor Lead Detection 6/55

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

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

  9. 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

  10. 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

  11. 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

  12. 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

  13. 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

  14. Lead Sensor The Amplifier Speaker: Jeff Hofmann 14/55

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

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

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

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

  19. More AHL = Less GFP! 19/55

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

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

  22. Lead Sensor Sequencing Speaker: Rohan Maddamsetti 22/55

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

  24. Tri-Stable Switch Speaker: Kyle Schutter 24/55

  25. 25/55

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

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

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

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

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

  31. 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

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

  33. 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

  34. 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

  35. 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

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

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

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

  39. Transformation Restriction Ligation The 3 Major Steps 39/55

  40. 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

  41. 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

  42. 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

  43. 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

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

  45. 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

  46. 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

  47. 47/55

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

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

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