1 / 55

Brown iGEM

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.

cala
Download Presentation

Brown iGEM

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related