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Reguli KAIST iGEM 2012 Regulation of E.coli Regulation of + - PowerPoint PPT Presentation

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Reguli KAIST iGEM 2012 Regulation of E.coli Regulation of + E.coli Regu Regu Regulation + E.coli li li Prototype Mycobacterium Phage Bxb1, gp35(DNA integrase) & gp47(DNA excisionase) Target att B att P Mycobacterium Phage

  1. Reguli KAIST iGEM 2012

  5. Regulation of E.coli

  7. Regulation of + E.coli

  8. Regu Regu Regulation + E.coli li li

  9. Prototype

  10. Mycobacterium Phage Bxb1, gp35(DNA integrase) & gp47(DNA excisionase) Target att B att P Mycobacterium Phage Bxb1 Integrase Target att L att R Mycobacterium Phage Bxb1 Integrase/Excisionase complex Target att B att P

  11. Prototype : Design RFP GFP att Promoter att P B GF P RFP GFP att L att R RFP

  12. Prototype : Experimental Data - Cell Culture

  13. Prototype : Experimental Data - Confocal Microscopy GFP initial + Integrase uninduced induced

  14. Prototype : Experimental Data - Fluorescent Intensity GFP initial 90000 140000 Induction (RFU) RFP(RFU) GFP(RFU) RFP(RFU) Induction GFP 6hr 12hr 6hr 12hr MG1655 negative control GFP initial + Integrase GFP control GFP initial + Integrase : Induced RFP control

  15. Prototype : Experimental Data - Fluorescent Intensity RFP initial 40000 16000 (RFU) RFP(RFU) GFP(RFU) RFP(RFU) Induction GFP Induction 6hr 12hr 6hr 12hr MG1655 negative control RFP initial + Integrase GFP control RFP initial + Integrase : Induced RFP control

  16. Reguli Module

  17. Reguli Module Reguli Module : Design

  18. Reguli Module : Design Bacterial Flavin-containing Monooxygenase Applicability Visibility N-acyl homoserine lactone Regulatory factor

  19. Reguli Module : Mechanism

  20. Reguli Module : Mechanism Terminator BBa_B0015 LuxR pLuxR pAutoIntegrase Promoter 5876bp BBa_J23119 Bxb1 gp35 (Int) pSB4A5

  21. Terminator BBa_B0015 LuxR LuxR pLuxR pAutoIntegrase Promoter 5876bp BBa_J23119 Bxb1 gp35 (Int) pSB4A5

  22. Promoter attP BBa_J23119 LuxI attB pAuto bFMO 5832bp pSB1C3 aiiA Bxb1 gp47 (Xis)

  23. LuxI Promoter attP BBa_J23119 LuxI attB pAuto bFMO 5832bp pSB1C3 aiiA Bxb1 gp47 (Xis)

  24. AHL LuxI Promoter attP BBa_J23119 LuxI attB pAuto bFMO 5832bp pSB1C3 aiiA Bxb1 gp47 (Xis)

  25. pAuto pAutoIntegrase 5832bp 5876bp LuxR LuxR AHL LuxI

  26. pAuto pAutoIntegrase 5832bp 5876bp LuxR LuxR AHL LuxI LuxR / AHL complex

  27. LuxR/AHL complex Terminator BBa_B0015 LuxR pLuxR pAutoIntegrase Promoter 5876bp BBa_J23119 Bxb1 gp35 (Int) pSB4A5

  28. LuxR/AHL complex Terminator BBa_B0015 LuxR pLuxR pAutoIntegrase Promoter 5876bp BBa_J23119 Bxb1 gp35 (Int) pSB4A5 Bxb1 gp35

  29. Bxb1 gp35 attP LuxI attB pAuto 5832bp bFMO pSB1C3 aiiA Bxb1 gp47 (Xis)

  30. Bxb1 gp35 attR attP LuxI attB attL pAuto 5832bp bFMO pSB1C3 aiiA Bxb1 gp47 (Xis)

  31. “BIO - INDIGO” Inverted Promoter attR Isatin attL pAuto bFMO 5832bp Indole bFMO

  32. attR attL pAuto bFMO 5832bp aiiA

  33. attR attL pAuto bFMO 5832bp Lactonase aiiA

  34. attR attL pAuto bFMO 5832bp aiiA

  35. pLuxR pAutoIntegrase 5876bp attR attL pAuto bFMO 5832bp Bxb1 gp47 (Xis) aiiA

  36. pLuxR pAutoIntegrase 5876bp attR attL pAuto bFMO 5832bp Bxb1 gp47 (Xis) aiiA Bxb1 gp47

  37. attR Bxb1 gp35 attL pAuto bFMO 5832bp Bxb1 gp47 (Xis) aiiA Bxb1 gp47

  38. attR Bxb1 gp35 Bxb1 gp35/47 complex attL pAuto bFMO 5832bp Bxb1 gp47 (Xis) aiiA

  39. attR attP Bxb1 gp35/47 complex Bxb1 gp35 attB attL pAuto bFMO 5832bp Bxb1 gp47 (Xis) aiiA

  40. Reguli Module : Vector Design Promoter Terminator BBa_J23119 BBa_B0015 attP LuxR attB LuxI pLuxR pAuto pAutoIntegrase Promoter 5832bp 5876bp BBa_J23119 bFMO pSB1C3 Bxb1 gp35 (Int) aiiA pSB4A5 Bxb1 gp47 (Xis) low copy number high copy number

  41. ATP NADH Conventional Methods Inducer Promoter synthetic endogenous Binding site CDS IPTG

  42. Reguli Module : Advantages No Inducer Autonomous Regulation ON IPTG OFF

  43. Reguli Module : Modeling

  44. Reguli Module : Modeling

  45. Reguli Module : Modeling - Prototype

  46. Reguli Module : Modeling - Prototype

  47. Reguli Module : Modeling - Prototype

  48. Reguli Module : Modeling - Prototype

  49. Reguli Module : Modeling - Prototype

  50. Reguli Module : Modeling - Reguli Module

  51. Reguli Module : Modeling - Reguli Module Conventional Reguli

  52. Reguli Module : Modeling - Reguli Module

  53. Reguli Module : Experimental Data

  54. Reguli Module : Experimental Data pAuto pAutoSimple 5832bp 4217bp 3kb 2kb 1kb pAutoIntegrase 500bp pAutoSimple pAuto 5876bp

  55. Reguli Module : Experimental Data Color change of host cells 0hr 3hr 6hr 9hr 15hr Inverted

  56. Reguli Module : Applications

  57. Reguli Module - Highly applicable att Promoter att P B

  58. Environmental Applications Bacteri-Guard

  59. Environmental Applications Bacteri-Guard Sensor- induced Bxb1 gp35 E.coli promoter (Int) Constitutive Genom attP Promoter e attB Pollutant sensor pBacteri-Guard Scaveng er vector GFP Bxb1 gp47 (Xis)

  60. Environmental Applications Bacteri-Guard Sensor- induced Bxb1 gp35 E.coli promoter (Int) Constitutive Genom attP Promoter e attB Pollutant sensor pBacteri-Guard Scaveng er vector GFP Bxb1 gp47 (Xis)

  61. Environmental Applications Bacteri-Guard Sensor- induced Bxb1 gp35 E.coli promoter (Int) Constitutive Genom attP Promoter e attB Pollutant sensor pBacteri-Guard Scaveng er vector GFP Bxb1 gp47 (Xis)

  62. Environmental Applications Bacteri-Guard Pollutant Pollutant sensor sensor pBacteri-Guard pBacteri-Guard Scaveng Scaveng er er vector vector GFP GFP Bxb1 gp47 (Xis) Bxb1 gp47 (Xis)

  63. Environmental Applications Bacteri-Guard Pollutant Pollutant sensor sensor pBacteri-Guard pBacteri-Guard Scaveng Scaveng er er vector vector GFP GFP Bxb1 gp47 (Xis) Bxb1 gp47 (Xis)

  64. Previous idea Bacteri-Guard pollutants pollutants

  65. Conclusion

  66. Conclusion In this project, we iGEM 2012 KAIST, Novel Biobrick parts Highly Manageable Biological System Highly Manageable Biological System Environmental Applications

  67. Human Practice JUL 24 th ~27 th , Talk with middle school students AUG 20 th , Talk with high school students AUG 6 th ~10 th , Talk with adults AUG 24 th , Talk with Experts(Biologists)

  68. THANK YOU

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