1. Davidson College Synth-Aces Tamar Odle (’08), Oscar Hernandez (’06), Kristen DeCelle (’06), Andrew Drysdale (’07), Matt Gemberling (’06), and Nick Cain (’06)

2. Long-term goal: detect eight different combinations of 3 chemicals Use RNA-mediated regulation of protein production Designed antiswitches, riboswitches, and external guide sequences. Overview of Digital Decoder Project

3. Human-readable output for 8 different combinations Inputs are detected by aptamers that have “on” or “off” switches Visualization of Decoder

4. One of our initial goals was to assemble 4 fluorescent proteins: EYFP, RFP, GFP, and ECFP. Measuring kinetics of proteins Degradation tags to inhibit the fluorescence of the proteins Oddities with I6060 in different strains of E. coli Parts still needed The Building Blocks

5. A Generic Fluorescent Protein Constitutive in some strains Repressed in some strains Inducible with IPTG Strongest degradation tag (LVA)

6. Building Necessary Parts

7. Streamlining Synthetic Biology Standardized DNA constructs Bio Brick Ends Limit the number of preparation steps “Plug and play” process

8. Our Plans Modular plasmid construction Save intermediate constructs Three RNA-mediated controls Kinetics of proteins +/- LVA

9. Our Intermediate Constructs Step 2 Step 3 Step 4

10. Inserting RNA Regulator

11. Characterization of Parts in Registry Part A Fluorescence Time Part B Fluorescence Time

12. Characterization of I6060

13. Initial Observations MC4100 DH5  I6060 in

14. Fluorescence Variations I6060 in DH5  (June 23) I6060 in DH5  (June 24) I6060 in MC4100 (June 26)

15. I6060 in 3 Cell Strains MC4100 DH5  JM109

16. Fluorescence of I6060 in 3 Cell Strains Fluorescence/Absorbance Time (Hours) July 26, 2005October 29-30, 2005

17. Why Does Fluorescence Vary? Hypothesis 1-Genetic variations between strains. Hypothesis 2-Genetic variations between cells. Hypothesis 3-Mutation in I6060 plasmid. Less protein is energetically favorable for cell. Hypothesis 4-Switch is flipped turning on proteases.

18. Engineering mRNA to Self-regulate

19. RNA Regulatory Molecules RNA regulation provides fast feedback Several viable approaches to RNA-mediated regulation

20. The Cis-acting Switches E. coli contain diverse array of natural riboswitches Our design used MTCT8-4 aptamer (Jenison, 1994) Previous E. coli riboswitches (Gallivan, 2004) Goals: minimize aptamer length test effects of aptamer position

21. General Riboswitch Design Lac promoter MCT8-4 aptamer RBS 8 bp spacer

22. Forward Riboswitch Construct Riboswitch ligated to EYFP ForAptRibo

23. Alternative Riboswitch Configurations 3AptRibo RevAptRibo

24. Riboswitch Activity Construct ForAptRibo RevAptRibo 3AptRibo I6060 Exp # 1 2

25. Creating Independent RNAs to Regulate Protein Production

26. Antiswitches Adapted to target EYFP (Smolke and Bayer, 2005) Regulate yeast protein translation Antiswitches tested; redesigns not synthesized yet

27. First Generation Antiswitchs “On” antiswitch appears to turn on EYFP “Off” antiswitch also appears to turn on EYFP Antiswitch targeting RBS not effective

28. Second Generation Antiswitchs Single plasmids Shared promoter

29. External Guide Sequences Trans-acting RNA “Off” EGS “On” EGS with aptamer?

30. EGS Plasmid

31. Digital Decoder Device Design

32. Goal: Decode combination of three chemicals, display 0-7 on a digital display Approach: Control display using antiswitches, responsive to: Theophylline Caffeine Malachite Green Digital Decoder Device

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

34. 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

35. 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

36. 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

37. 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

38. 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

39. 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

40. 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

41. 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

42. 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

43. 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

44. Two different antiswitches: “Off” antiswitch: Suppress expression of EYFP in the presence of its ligand “On” antiswitch: Allow expression of EYFP in the presence of its ligand

45. Inside E. coli “6”: 000000 Start Codon Promoter Theophylline Caffeine Malachite Green EYFP coding region

46. Inside E. coli “6”: Start Codon Promoter 010010 Theophylline Caffeine Malachite Green EYFP coding region

47. Start Codon Promoter 010010 Theophylline Caffeine Malachite Green Inside E. coli “6”: EYFP coding region

48. Inside E. coli “6”: Start Codon Promoter 010010 Theophylline Caffeine Malachite Green EYFP coding region

49. Inside E. coli “6”: Start Codon Promoter 010010 Theophylline Caffeine Malachite Green EYFP coding region

50. Inside E. coli “6”: Start Codon Promoter 010010 Theophylline Caffeine Malachite Green EYFP coding region

51. Inside E. coli “6”: YFP coding region Start Codon Promoter 010010 Theophylline Caffeine Malachite Green EYFP coding region

52. Inside E. coli “6”: EYFP coding region Start Codon Promoter 010010 Theophylline Caffeine Malachite Green

53. Inside E. coli “6”: Start Codon Promoter 010010 Theophylline Caffeine Malachite Green EYFP coding region

54. Inside E. coli “6”: Start Codon Promoter 110110 Theophylline Caffeine Malachite Green EYFP coding region

55. Inside E. coli “6”: Start Codon Promoter 110110 Theophylline Caffeine Malachite Green EYFP coding region

56. Inside E. coli “6”: Start Codon Promoter 110110 Theophylline Caffeine Malachite Green EYFP coding region

57. Start Codon Promoter 110110 Theophylline Caffeine Malachite Green Inside E. coli “6”: EYFP coding region

58. Inside E. coli “6”: Start Codon Promoter 110110 Theophylline Caffeine Malachite Green EYFP coding region

59. Start Codon Promoter Theophylline Caffeine Malachite Green 110110 Inside E. coli “6”: EYFP coding region

60. Inside E. coli “6”: Start Codon Promoter 111111 Theophylline Caffeine Malachite Green EYFP coding region

61. Inside E. coli “6”: Start Codon Promoter 111111 Theophylline Caffeine Malachite Green EYFP coding region

62. Inside E. coli “6”: Start Codon Promoter 111111 Theophylline Caffeine Malachite Green EYFP coding region

63. Inside E. coli “6”: Start Codon Promoter 111111 Theophylline Caffeine Malachite Green EYFP coding region

64. Inside E. coli “6”: Start Codon Promoter 111111 Theophylline Caffeine Malachite Green EYFP coding region

65. Inside E. coli “6”: Start Codon Promoter 111111 Theophylline Caffeine Malachite Green EYFP coding region

66. “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

67. Thanks to MIT hosts and iGEM organizers Funding support from: HHMI, NIH via MIT, Duke Endowment, and Davidson College