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. “Digital Decoder Device” A F G B C D E E. Coli strain Strips in Decoder Theophyllin e CaffeineMalachite Green 0ABCDEF000 1CD001 2BCDEFG010 3BCGDE011 4AGCD100 5ABGDE101 6ABGDEF110 7BCD111

7. A F G B C D E 0 0 0 0 0 0 E. Coli strain Strips in Decoder TheophyllineCaffeineMalachite Green 0ABCDEF000 1CD001 2BCEFG010 3BCGDE011 4AGCD100 5ABGDE101 6AGDEF110 7BCD111 Digital Decoder Device

8. A F G B C D E 0 0 0 0 0 0 1 1 E. Coli strain Strips in Decoder TheophyllineCaffeineMalachite Green 0ABCDEF000 1CD001 2BCEFG010 3BCGDE011 4AGCD100 5ABGDE101 6AGDEF110 7BCD111 Digital Decoder Device

9. A F G B C D E 0 0 0 0 0 0 1 1 2 2 2 2 2 E. Coli strain Strips in Decoder TheophyllineCaffeineMalachite Green 0ABCDEF000 1CD001 2BCEFG010 3BCGDE011 4AGCD100 5ABGDE101 6AGDEF110 7BCD111 Digital Decoder Device

10. A F G B C D E 0 0 0 0 0 0 1 1 2 2 2 2 2 3 3 3 3 3 E. Coli strain Strips in Decoder TheophyllineCaffeineMalachite Green 0ABCDEF000 1CD001 2BCEFG010 3BCGDE011 4AGCD100 5ABGDE101 6AGDEF110 7BCD111 Digital Decoder Device

11. A F G B C D E 0 0 0 0 0 0 1 1 2 2 2 2 2 3 3 3 3 3 4 4 4 4 E. Coli strain Strips in Decoder TheophyllineCaffeineMalachite Green 0ABCDEF000 1CD001 2BCEFG010 3BCGDE011 4AGCD100 5ABGDE101 6AGDEF110 7BCD111 Digital Decoder Device

12. A F G B C D E 0 0 0 0 0 0 1 1 2 2 2 2 2 3 3 3 3 3 4 4 4 4 5 5 5 5 5 E. Coli strain Strips in Decoder TheophyllineCaffeineMalachite Green 0ABCDEF000 1CD001 2BCEFG010 3BCGDE011 4AGCD100 5ABGDE101 6AGDEF110 7BCD111 Digital Decoder Device

13. A F G B C D E 0 0 0 0 0 0 1 1 2 2 2 2 2 3 3 3 3 3 4 4 4 4 5 5 5 5 5 6 6 6 6 6 E. Coli strain Strips in Decoder TheophyllineCaffeineMalachite Green 0ABCDEF000 1CD001 2BCEFG010 3BCGDE011 4AGCD100 5ABGDE101 6AGDEF110 7BCD111 Digital Decoder Device

14. A F G B C D E 0 0 0 0 0 0 1 1 2 2 2 2 2 3 3 3 3 3 4 4 4 4 5 5 5 5 5 6 6 6 6 6 7 7 7 E. Coli strain Strips in Decoder TheophyllineCaffeineMalachite Green 0ABCDEF000 1CD001 2BCEFG010 3BCGDE011 4AGCD100 5ABGDE101 6AGDEF110 7BCD111 Digital Decoder Device

15. A F G B C D E 0 0 0 0 0 0 1 1 2 2 2 2 2 3 3 3 3 3 4 4 4 4 5 5 5 5 5 6 6 6 6 6 7 7 E. Coli strain Strips in Decoder TheophyllineCaffeineMalachite Green 0ABCDEF000 1CD001 2BCEFG010 3BCGDE011 4AGCD100 5ABGDE101 6AGDEF110 7BCD111 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 7

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”: 000000 Start Codon Theophylline Caffeine Malachite Green YFP mRNA 5’ UTR

18. Inside E. coli “6”: Start Codon Theophylline Caffeine Malachite Green 010010 YFP mRNA 5’ UTR

19. Inside E. coli “6”: YFP mRNA Start Codon Theophylline Caffeine Malachite Green 010010 5’ UTR

20. Inside E. coli “6”: Start Codon Theophylline Caffeine Malachite Green 010010 YFP mRNA 5’ UTR

21. Inside E. coli “6”: Start Codon Theophylline Caffeine Malachite Green 010010 YFP mRNA 5’ UTR

22. Inside E. coli “6”: Start Codon Theophylline Caffeine Malachite Green 010010 YFP mRNA 5’ UTR

23. Inside E. coli “6”: YFP coding region Start Codon Theophylline Caffeine Malachite Green 010010 YFP mRNA 5’ UTR

24. Inside E. coli “6”: Start Codon Theophylline Caffeine Malachite Green 010010 YFP mRNA 5’ UTR

25. Inside E. coli “6”: Start Codon Theophylline Caffeine Malachite Green 010010 YFP mRNA 5’ UTR

26. Inside E. coli “6”: Start Codon Theophylline Caffeine Malachite Green 110110 YFP mRNA 5’ UTR

27. Inside E. coli “6”: Start Codon Theophylline Caffeine Malachite Green 110110 YFP mRNA 5’ UTR

28. Inside E. coli “6”: Start Codon Theophylline Caffeine Malachite Green 110110 YFP mRNA 5’ UTR

29. Inside E. coli “6”: Start Codon Theophylline Caffeine Malachite Green 110110 YFP mRNA 5’ UTR

30. Inside E. coli “6”: Start Codon Theophylline Caffeine Malachite Green 110110 YFP mRNA 5’ UTR

31. Inside E. coli “6”: Start Codon Theophylline Caffeine Malachite Green 111111 YFP mRNA 5’ UTR

32. Inside E. coli “6”: Start Codon Theophylline Caffeine Malachite Green 111111 YFP mRNA 5’ UTR

33. Inside E. coli “6”: Start Codon Theophylline Caffeine Malachite Green 111111 YFP mRNA 5’ UTR

34. Inside E. coli “6”: Start Codon Theophylline Caffeine Malachite Green 111111 YFP mRNA 5’ UTR

35. Inside E. coli “6”: Start Codon Theophylline Caffeine Malachite Green 111111 YFP mRNA 5’ UTR

36. Inside E. coli “6”: Start Codon Theophylline Caffeine Malachite Green 111111 YFP mRNA 5’ UTR

37. Inside E. coli “6”: Start Codon Theophylline Caffeine Malachite Green 111111 5’ UTR YFP mRNA

38. “Digital Decoder Device” A F G B C D E 0 0 0 0 0 0 1 1 2 2 2 2 2 3 3 3 3 3 4 4 4 4 5 5 5 5 5 6 6 6 6 6 7 7 7 E. coli strain Strips in Decoder TheophyllineCaffeineMalachite Green 0ABCDEF000 1CD001 2BCEFG010 3BCGDE011 4AGCD100 5ABGDE101 6AGDEF110 7BCD111

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 Theophylline Theophylline aptamer Caffeine

42. Ribozymes

44. How do you build antiswitches?

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

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

47. Blue Heron

48. BioBrick prefixes and suffixes

49. Mixed Site

53. Antiswitch Insert

54. Results Verify construction Terminator + YFP Add antiswitch On Off

55. Antiswitch Testing E. coli + antiswitch + theophylline Measure cell density Measure fluorescence Divide fluorescence by cell density

56. ‘Off’ antiswitch ‘On’ antiswitch

57. Antiswitch v2.0 Antiswitches were uncontrollable One problem –No space between the promoter and first ribozyme –E. coli require 5 to 9 base pair space –PCR to add these base pairs

59. Results v2.0 Verify construction PCR products

60. Results v2.0 PCR products + Promoter ONOFF

61. Results E. coli + antiswitch + theophylline Time course measurements

68. Conclusions Antiswitches don’t function in prokaryotes Pursue alternative mechanisms

69. Future Research Issues with antiswitches –Stoicheometry issues –Ribozyme cleavage –Predicted vs. actual folding of antiswitch –RNA stability –Rate of initiation

70. Continue with Antiswitches?

71. Future of Synthetic Biology Very appropriate for undergraduates More Ph.D. degrees Imagination and creativity encouraged –Malaria medicine

72. Acknowledgements Collaborators –O. Hernandez, T. Odle, K. DeCelle, N. Cain, A. Drysdale, M. Cowell, J. Ryan, F. Trappey, Dr. A. M. Campbell, Dr. L. Heyer, Dr. K. Bernd, Dr. D. Wessner, Davidson College –Dr. D. Endy and R. Rettberg, MIT –T. Bayer, CalTech Financial support from HHMI, MIT, and Davidson Biology Dept.

73. External Guide Sequences