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Synthetic Biology: Design and Characterization of antiswitches in E .coli

Synthetic Biology: Design and Characterization of antiswitches in E .coli. Matt Gemberling 27 April 2006. Outline . Overview of Synthetic Biology Introduce antiswitches Construct antiswitches Results The future of prokaryotic antiswitches. Synthetic Biology. Caffeine Detector.

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Synthetic Biology: Design and Characterization of antiswitches in E .coli

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  1. Synthetic Biology: Design and Characterization of antiswitches in E .coli Matt Gemberling 27 April 2006

  2. Outline • Overview of Synthetic Biology • Introduce antiswitches • Construct antiswitches • Results • The future of prokaryotic antiswitches

  3. Synthetic Biology

  4. Caffeine Detector

  5. “Digital Decoder Device” Goal: Given a combination of three chemicals, represent the numbers 0-7 on a digital display Approach: Control fluorescence of display components using antiswitches, responsive to: Theophylline Caffeine Malachite Green

  6. B C A G F D E “Digital Decoder Device”

  7. Digital Decoder Device B 0 0 0 C A G 0 0 F D 0 E

  8. Digital Decoder Device B 0 0 0 1 C A G 0 0 1 F D 0 E

  9. Digital Decoder Device B 0 2 0 0 1 2 C A G 2 0 0 2 1 F D 0 2 E

  10. Digital Decoder Device B 0 2 3 0 0 1 2 C 3 A G 2 3 0 0 2 1 3 F D 0 2 3 E

  11. Digital Decoder Device B 0 2 3 0 0 4 1 2 C 3 A 4 G 2 3 4 0 0 2 1 3 F 4 D 0 2 3 E

  12. Digital Decoder Device B 0 2 3 5 0 0 4 1 5 2 C 3 A 4 G 2 3 4 5 0 0 2 1 3 F 4 D 5 0 2 3 5 E

  13. Digital Decoder Device B 0 2 3 5 0 0 4 1 5 2 C 6 3 A 4 G 6 2 3 4 5 0 0 2 1 6 3 F 4 D 5 6 6 0 2 3 5 E

  14. Digital Decoder Device B 0 2 3 5 7 0 0 4 1 5 2 C 6 3 A 4 7 G 6 2 3 4 5 0 0 2 1 6 3 F 4 D 5 6 7 6 0 2 3 5 E

  15. Digital Decoder Device B 0 2 3 5 7 0 1 6 2 C 0 3 A 4 4 5 7 G 6 2 3 4 5 0 • Multiple strains live in each strip of the decoder • Only one strain will fluoresce at any given time, and it will illuminate its entire strip. • A particular strain will only fluoresce when its corresponding chemical combination is present • Combinations of “on” and “off” antiswitches required 1 6 3 F 0 4 D 2 5 6 7 6 0 2 3 5 E

  16. Two different antiswitches: “Off” antiswitch: Will suppress expression of YFP in the presence of its ligand “On” antiswitch: Will allow expression of YFP in the presence of its ligand

  17. Inside E.coli “6”: 0 0 0 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon

  18. Inside E.coli “6”: 0 1 0 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon

  19. Inside E. coli “6”: 0 1 0 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon

  20. Inside E.coli “6”: 0 1 0 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon

  21. Inside E.coli “6”: 0 1 0 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon

  22. Inside E.coli “6”: 0 1 0 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon

  23. YFP coding region Inside E.coli “6”: 0 1 0 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon

  24. Inside E.coli “6”: 0 1 0 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon

  25. Inside E.coli “6”: 0 1 0 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon

  26. Inside E.coli “6”: 1 1 0 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon

  27. Inside E.coli “6”: 1 1 0 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon

  28. Inside E.coli “6”: 1 1 0 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon

  29. Inside E. coli “6”: 1 1 0 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon

  30. Inside E.coli “6”: 1 1 0 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon

  31. Inside E. coli “6”: 1 1 1 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon

  32. Inside E.coli “6”: 1 1 1 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon

  33. Inside E.coli “6”: 1 1 1 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon

  34. Inside E.coli “6”: 1 1 1 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon

  35. Inside E.coli “6”: 1 1 1 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon

  36. Inside E.coli “6”: 1 1 1 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon

  37. Inside E.coli “6”: 1 1 1 Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR Start Codon

  38. “Digital Decoder Device” B 7 0 2 3 5 0 1 6 2 C 0 3 A 4 4 5 7 G 6 2 3 4 5 0 1 6 3 F 0 4 D 2 5 6 7 6 0 2 3 5 E

  39. RNA Antiswitches • Smolke and Bayer (2005) first antiswitches in yeast (S. cerevisiae) • “On” and “Off” switches were designed • RNA-mediated regulation of translation

  40. Antiswitch (RNA)

  41. Aptamer • Nucleic acid (e.g., RNA) • Binds specific ligand Caffeine Theophylline Theophylline aptamer

  42. Ribozymes

  43. How do you build antiswitches?

  44. Construction of Antiswitches • Registry of Standard Biological Parts (http://parts.mit.edu) • Synthesize de novo

  45. Registry • Offers 2,100 parts • Standardized • Modular • Data available for most parts • Characterization • Sequences • Updated in real-time (wiki)

  46. Blue Heron

  47. BioBrick prefixes and suffixes

  48. Mixed Site

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