1. Endy, BIOS, June 2004 1 Atrophy time SRM Physical Performance (short) SRM Adapt & Learn (medium) SRM Evolve or Persist (long) SRM Encoding Design of Self-Replicating Machines

2. Endy, BIOS, June 2004 2 Yeast Consider a Cell Signaling Pathway Cell Cycle

3. Endy, BIOS, June 2004 3 Yeast Cell Cycle Consider a Different Cell Signaling Pathway Budding Metabolism Yet Another Signaling Pathway

4. Endy, BIOS, June 2004 4

5. 5 Atrophy SRM T7 Encoding What does T7 know?

6. Endy, BIOS, June 2004 6 F. William Studier, Science 176:403 (1972) Deletion Amber/Enzyme Amber/Essential

7. Endy, BIOS, June 2004 7 Previous Page Sequence (BNL) Dunn & Studier, J. Mol. Bio. 166:477 (1983)

8. Endy, BIOS, June 2004 8 Atrophy SRM T7 What We Know What We Need To Know Encoding What do we know?

9. Endy, BIOS, June 2004 9 Atrophy What We Make SRM T7-SB Encoding Genome Domestication

10. Endy, BIOS, June 2004 10 A0 TR/ SRL ø OLA1A2 R0.3 BoxA 0.30.40.5R0.50.6A/B0.7 C R11 ø 1.1A ø 1.1B R1.11.11.2 ø 1.3 R1.31.3TE1.41.51.61.7 ø 1.5 ø 1.6 A2 NsiIPciIMfeISpeIBclI B

11. Endy, BIOS, June 2004 11 A0 63 TR/ SRL ø OL 35 A1 88 A2 316 R0.3 BoxA 0.3 370 0.4 183 0.5 158 R0.5 52 0.6A/B 355 0.7 110291 C R11 2708139 ø 1.1A ø 1.1B R1.11.1 148 1.2 27580 ø 1.3 R1.31.3 1099 TE 65 1.4 173 1.5 110 1.6 279 1.7 164 ø 1.5 35 ø 1.6 35 A2 210443 NsiIPciIMfeISpeIBclI

12. Endy, BIOS, June 2004 12 A0 63 TR/ SRL ø OL 35 A1 88 A2 316 R0.3 BoxA 0.3 370 0.4 183 0.5 158 R0.5 52 0.6A/B 355 0.7 110291 C R11 2708139 ø 1.1A ø 1.1B R1.11.1 148 1.2 27580 ø 1.3 R1.31.3 1099 TE 65 1.4 173 1.5 110 1.6 279 1.7 164 ø 1.5 35 ø 1.6 35 A2 210443 NsiIPciIMfeISpeIBclISacINheIApaLISapI

13. Endy, BIOS, June 2004 13

14. Endy, BIOS, June 2004 14

15. Endy, BIOS, June 2004 15 T7 + Rebuild [1-8311] :T7 +

16. Endy, BIOS, June 2004 16

17. Endy, BIOS, June 2004 17 D23L AvrII D24L EcoRI D26L BamHI D27L EagI D28L SacII D30L SalI D22L BstEII D22RD23RD24RD26RD27RD28RD30R ø2.5 32.8 ø3.8 3.53.8R3.8 D25L XmaI D25R 2.5 D29L PciI D29R 1.82 BglII 175209357234384644787530675 FspIAvrII D50L BsiWI D51L PvuI D53L SacII D54L BamHI D55L XmaI D57L HindIII D50RD51RD53RD54RD55RD57R ø9 10ATøTø12 D52L EcoRI D52R 9 D56L ApaI D56R 8 PacI 1611351038354647359123823 ø10 11 DraIRsrII XbaI BssHIIAatIIAvaIBspHIEciINsiI NdeI AseI D57L HindIII D59L EcoRI D58L PfoI D57R D58R 1213 PacI 2382624173 ø13 R13 D60L BsiWI D62L EagI D59RD60RD62R 16 D61L PvuI D61R 15 ApaLI 59122443520437 14 NciI ScaI BstBIAvaI D64L HindIII D65L BamHI D67L ApaI D68L EcoRI D69L BsiWI D71L SalI D63L SacII D63RD64RD65RD67RD68RD69RD71R ø17 18.5/18.718 E 19/19.2/19.319.5R18.5 D66L XmaI D66R 17.5 D70L PstI D70R 17 35166220427053460176135150 øOR SRR/TR 160 KasIBspDIFspI BglI BlpIDraIIIAcvIAciIKpnI

18. Endy, BIOS, June 2004 18

19. Endy, BIOS, June 2004 19 E.colibrator, Polkadorks @ IAP 2004

20. Endy, BIOS, June 2004 20 Application Struggle, Limited Success, Struggle… System?? Devices? Design & Fabrication

21. Endy, BIOS, June 2004 21 Application Struggle, Success, Predictable Success Systems Parts & Fabrication Design Applications Tools Registries Synthesis Measurement

22. Endy, BIOS, June 2004 22 Standardization of Components –Predictable performance –Off-the-shelf –ME, 1800s Abstraction –Insulate relevant characteristics from overwhelming detail –Simple artifacts that can be used in combination –From Physics to EE, 1900s Decoupling Design & Fabrication –Rules insulating design process from details of fabrication –Enable parts, device, and system designers to work together –VLSI electronics, 1970s Enabling Biological Engineering

23. Endy, BIOS, June 2004 23 Abstraction Hierarchy Devices Parts Systems

24. Endy, BIOS, June 2004 24 Parts Zif268, Paveltich & Pabo c. 1991

25. Endy, BIOS, June 2004 25 Devices cI-857 O Lac RBS T CI LacI CI

26. Endy, BIOS, June 2004 26 Devices LacI  CI inverter CI LacI

27. Endy, BIOS, June 2004 27 Systems Inverter.2Inverter.3Inverter.1

28. Endy, BIOS, June 2004 28 Interfaces Devices Parts Systems Inv.2Inv.3Inv.1 Zif268, Paveltich & Pabo c. 1991 LacI  CI inverter CI LacI

29. Endy, BIOS, June 2004 29 Parts/Device Interface Devices Parts Zif268, Paveltich & Pabo c. 1991 LacI  CI inverter CI LacI

30. Endy, BIOS, June 2004 30 Stories “In 1910, I was in Mexico, in the state of Yucatan, when an invasion of locusts occured; the Indians reported to me that in a certain place the ground was strewn with the corpses of these insects. I went there and collected sick locusts, easily picked out since their principal symptom was an abundant blackish diarrhoea. This malady had not as yet been described, so I studied it. It was a septicaemia with intestinal symptoms, It was caused by bacteria, the locust coccobacilli, which were present almost in the pure state in the diarrhoeal liquid. I could start epidemics in columns of healthy insects by dusting cultures of the coccobacillus on plants in front of the advancing columns: the insects infected themselves as they devoured the soiled plants… In the course of these researches, at various times I noticed an anomaly, shown by some cultures of the coccocacillus which intrigued me greatly, although in fact the observation was ordinary enough., so banal indeed that many bacteriologists had certainly made it before on a variety of cultures. The anomaly consisted of clear spots, quite circular, two or three millimeters in diameter, speckling the cultures grown on agar.” -- The Bacteriophage by Dr. Felix d'Herelle, Science News 14: 44-59 (1949). (Translation by J. L. Crammer)

31. Endy, BIOS, June 2004 31 Parts/Device Interface Devices Parts Zif268, Paveltich & Pabo c. 1991 LacI  CI inverter CI LacI XX A  B inverter B A

32. Endy, BIOS, June 2004 32 Device/System Interface Devices Systems Inv.2Inv.3Inv.1 A  B inverter B A C  D inverter D C E  F inverter F E

33. Endy, BIOS, June 2004 33 Device/System Interface Devices Systems E  FC  DA  B inverter B A C  D inverter D C E  F inverter F E X X X

34. Endy, BIOS, June 2004 34 Device/System Interface Devices Systems E  FC  DA  B inverter B A C  D inverter D C E  F inverter F E A  D X X X

35. Endy, BIOS, June 2004 35 Device/System Interface cI-857 O Lac RBS T cI LacI cI-857 RBS T cI O PoPS in PoPS out LacI cI PoPS out PoPS in

36. Endy, BIOS, June 2004 36 cI RBS T O PoPS OUT PoPS IN cI PoPS OUT INVERTER PoPS OUT PoPS IN Polymerase Per Second = PoPS! PoPS OUT PoPS Source (Any)

37. Endy, BIOS, June 2004 37 PoPS OUT PoPS IN.1 PoPS  PoPS 2-input NOR PoPS IN.2 cI RBS T O CI PoPS OUT PoPS IN.1 cI RBS T PoPS IN.2 PoPS Fan-In

38. Endy, BIOS, June 2004 38 cI RBS T O CI PoPS OUT.1 PoPS IN T O PoPS OUT.2 PoPS OUT.1 PoPS IN PoPS OUT.2 PoPS  PoPS 2-output NOT PoPS Fan-Out

39. Endy, BIOS, June 2004 39 PoPS-based Composition PoPS OUT PoPS IN.1 PoPS  PoPS 2-input NOR PoPS IN.2 PoPS OUT.1 PoPS IN PoPS OUT.2 PoPS  PoPS 2-output NOT

40. Endy, BIOS, June 2004 40 Device/System Interface Devices Systems X A  B inverter B A C  D inverter D C E  F inverter F E

41. Endy, BIOS, June 2004 41 Device/System Interface Devices Systems BCA A B C PoPS IN PoPS OUT X

42. Endy, BIOS, June 2004 42 Interfaces Devices Parts Systems PoPS Inv.2 PoPS Inv.3 PoPS Inv.1 Zif268, Paveltich & Pabo c. 1991 PoPS Inv.1 PoPS

43. Endy, BIOS, June 2004 43 E.colibrator, Polkadorks @ IAP 2004

44. Endy, BIOS, June 2004 44 Device-Level System Diagram

45. Endy, BIOS, June 2004 45 Parts- and Device-Level System Diagram

46. Endy, BIOS, June 2004 46 DNA Layout

47. Endy, BIOS, June 2004 47 Population-Level Simulations

48. Endy, BIOS, June 2004 48 Trigger Test Circuit Characterization and Debug

49. Endy, BIOS, June 2004 49 MIT Registry of Standard Biological Parts http://parts.mit.edu/

50. Endy, BIOS, June 2004 50 DNA Synthesis Year Bases of DNA Per Person Day Carlson, Pace & Proliferation of Biological Technologies, Biosec. & Bioterror. 1(3):1 (2003)

51. Endy, BIOS, June 2004 51 Hacking the Living World?

52. Endy, BIOS, June 2004 52 Risk Biological Risk: Background Technology Classes Relevant to Biological Risk (current relative capabilities) Manipulation Analysis Response Detection

53. Endy, BIOS, June 2004 53 Biological Risk: Tactics as “Strategy” Maginot Line France, 1940 Ciprofloxacin Smallpox vaccine Anthrax vaccine SARS assay VHF therapy (under construction) Plague vaccine (under construction)

54. Endy, BIOS, June 2004 54 Biological Risk: Future Strategy Risk Technology Classes Relevant to Future Biological Risk (needed capabilities) Detection Analysis Response Manipulation

55. Endy, BIOS, June 2004 55 Biological Risk: Suite of Solutions Number of Individuals Individual’s Intent honorable dishonorable Bin Laden Genetics, Inc. Disgruntled Researcher Garage Bio-Hacker Basic Researcher

56. Endy, BIOS, June 2004 56 A Constructive Society

57. Endy, BIOS, June 2004 57 D23L AvrII D24L EcoRI D26L BamHI D27L EagI D28L SacII D30L SalI D22L BstEII D22RD23RD24RD26RD27RD28RD30R ø2.5 32.8 ø3.8 3.53.8R3.8 D25L XmaI D25R 2.5 D29L PciI D29R 1.82 BglII 175209357234384644787530675 FspIAvrII D50L BsiWI D51L PvuI D53L SacII D54L BamHI D55L XmaI D57L HindIII D50RD51RD53RD54RD55RD57R ø9 10ATøTø12 D52L EcoRI D52R 9 D56L ApaI D56R 8 PacI 1611351038354647359123823 ø10 11 DraIRsrII XbaI BssHIIAatIIAvaIBspHIEciINsiI NdeI AseI D57L HindIII D59L EcoRI D58L PfoI D57R D58R 1213 PacI 2382624173 ø13 R13 D60L BsiWI D62L EagI D59RD60RD62R 16 D61L PvuI D61R 15 ApaLI 59122443520437 14 NciI ScaI BstBIAvaI D64L HindIII D65L BamHI D67L ApaI D68L EcoRI D69L BsiWI D71L SalI D63L SacII D63RD64RD65RD67RD68RD69RD71R ø17 18.5/18.718 E 19/19.2/19.319.5R18.5 D66L XmaI D66R 17.5 D70L PstI D70R 17 35166220427053460176135150 øOR SRR/TR 160 KasIBspDIFspI BglI BlpIDraIIIAcvIAciIKpnI

58. Endy, BIOS, June 2004 58 Kuroda-Kawaguchi et al., Nature Genetics 29:279 (2001)

59. Endy, BIOS, June 2004 59 Responsibility “Men have forgotten this truth,” said the fox. “But you must not forget it. You become responsible, forever, for what you have tamed.” Saint-Exupéry, Le Petit Prince (1943)

60. Endy, BIOS, June 2004 60 Acknowledgements Lab: Jennifer Braff, Barry Canton, Leon Chan, Caitlin Conboy, Jeffrey Gritton, Alison Hearn, Heather Keller, Jason Kelly, Sriram Kosuri, Maia Mahoney, Sara Neves, Sylvain Olier, Jose Pacheco, Francois St-Pierre, Samantha Sutton, Ilya Sytchev, and Ty Thomson Others: Adam Arkin, Frances Arnold, Ralph Baric, Roger Brent, Jehoshua Bruck, Carlos Bustamante, Rob Carlson, Austin Che, Jim Collins, Lynn Conway, Ron Davis, Mita Desai, Eric Eisenstadt, Michael Elowitz, Felice Frankel, Stephanie Forrest, Timothy Gardner, Seth Goldstein, Homme Hellinga, George Homsy, Joe Jacobsen, Tom Kalil, Jay Keasling, Doug Kirkpatrick, Tom Knight, Sri Kosuri, Patrick Lincoln, John Mulligan, Richard Murray, Radhika Nagpal, Richard Newton, Carl Pabo, Randy Rettberg, Pamela Silver, Brad Smith, Christina Smolke, Gerry Sussman, Samantha Sutton, Claire Tomlin, Jeffrey Way, Chris Webb, Ron Weiss, Scot Wolfe, Aarne Vesilind, the members of the MIT Synthetic Biology Working Group, and the students of the 2003 and 2004 MIT IAP Synthetic Biology Labs