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Atrophy

Encoding. SRM. SRM. SRM. Physical Performance (short). Adapt & Learn (medium). Evolve or Persist (long). Atrophy. Design of Self-Replicating Machines. SRM. time. Consider a Cell. Signaling Pathway. Yeast. Cell Cycle. Signaling Pathway. Consider a Different Cell. Yeast.

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Atrophy

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

  2. Consider a Cell Signaling Pathway Yeast Cell Cycle

  3. Signaling Pathway Consider a Different Cell Yeast Yet Another Signaling Pathway Budding Cell Cycle Metabolism

  4. Encoding Atrophy What does T7 know? SRMT7

  5. Amber/Essential Amber/Enzyme Deletion F. William Studier, Science176:403 (1972)

  6. Previous Page Sequence (BNL) Dunn & Studier, J. Mol. Bio.166:477 (1983)

  7. Encoding Atrophy What do we know? SRMT7 What We Need To Know What We Know

  8. Encoding Atrophy Genome Domestication What We Make SRMT7-SB

  9. PciI BclI NsiI MfeI SpeI ø1.6 øOL A1 A2 A2 C ø1.1A ø1.1B ø1.3 ø1.5 B BoxA TR/ SRL R0.3 0.3 0.4 R0.5 0.5 0.6A/B 0.7 R1 1 R1.1 1.1 1.2 R1.3 1.3 TE 1.4 1.5 1.6 1.7 A0

  10. BclI PciI NsiI MfeI SpeI 63 35 210 35 88 316 370 183 52 158 355 1102 91 2708 139 148 275 80 1099 65 173 35 110 279 164 443 ø1.6 øOL A1 A2 A2 C ø1.1A ø1.1B ø1.3 ø1.5 TR/ SRL BoxA R0.3 0.3 0.4 R0.5 0.5 0.6A/B 0.7 R1 1 R1.1 1.1 1.2 R1.3 1.3 TE 1.4 1.5 1.6 1.7 A0

  11. PciI BclI SacI NheI NsiI MfeI ApaLI SpeI SapI 63 35 210 35 88 316 370 183 52 158 355 1102 91 2708 139 148 275 80 1099 65 173 35 110 279 164 443 ø1.6 øOL A1 A2 A2 C ø1.1A ø1.1B ø1.3 ø1.5 TR/ SRL BoxA R0.3 0.3 0.4 R0.5 0.5 0.6A/B 0.7 R1 1 R1.1 1.1 1.2 R1.3 1.3 TE 1.4 1.5 1.6 1.7 A0

  12. D2L SphI D1L BstEII D20L XbaI D3L BspDI D4L HindIII D5L BssHII D6L SexAI D7L MluI D8L BsiWI D9L RsrII D9.5L SacII D10L EagI D10.5L PacI D11L EcoRI D12L PfoI D13L EcoO1091 D14L XmaI D15L ApaI D16L NcoI D17L KasI D18L AvrII D19L AatII D21L AgeI D1R D2R D3R D4R D5R D6R D7R D8R D9R D9.5R D10R D10.5R D11R D12R D13R D14R D15R D16R D17R D18R D19R D20R D21R SacI NheI NsiI PciI MfeI ApaLI SpeI SapI BclI 63 35 210 35 88 316 370 183 52 158 355 1102 91 2708 139 148 275 80 1099 65 173 35 110 279 164 443 ø1.6 øOL A1 A2 A2 C ø1.1A ø1.1B ø1.3 ø1.5 TR/ SRL BoxA R0.3 0.3 0.4 R0.5 0.5 0.6A/B 0.7 R1 1 R1.1 1.1 1.2 R1.3 1.3 TE 1.4 1.5 1.6 1.7 A0

  13. Rebuild[1-8311]:T7+ T7+

  14. D23L AvrII D24L EcoRI D25L XmaI D26L BamHI D27L EagI D28L SacII D29L PciI D30L SalI D22L BstEII D22R D23R D24R D25R D26R D27R D28R D29R D30R BglII 175 209 35 723 438 464 478 75 306 75 ø3.8 ø2.5 1.8 2 2.8 3 3.5 R3.8 3.8 2.5 D2L SphI D1L BstEII D20L XbaI D3L BspDI D4L HindIII D5L BssHII D6L SexAI D7L MluI D8L BsiWI D9L RsrII D9.5L SacII D10L EagI D10.5L PacI D11L EcoRI D12L PfoI D13L EcoO1091 D14L XmaI D15L ApaI D16L NcoI D17L KasI D18L AvrII D19L AatII D21L AgeI D1R D2R D3R D4R D5R D6R D7R D8R D9R D9.5R D10R D10.5R D11R D12R D13R D14R D15R D16R D17R D18R D19R D20R D21R AseI NsiI DraI BstEII BspHI EciI AvaI SpeI ScaI XcaI XbaI KpnI AcvI DraIII BlpI FspI AciI BspDI KasI D32L EagI D33L AscI D34L SgrI D35L HindIII D36L PfoI D37L NheI D38L SphI D39L NcoI D40L BamHI D41L SacII D42L ApaI D43L XmaI D44L SacI D45L XhoI D46L EcoRI D47L PvuI D48L RsrII D49L PstI D31L BsiWI SacI NheI NsiI PciI MfeI ApaLI SpeI SapI BclI D31R D32R D33R D34R D35R D36R D41R D44R D49R D37R D38R D39R D40R D42R D43R D45R D46R D47R D48R 63 35 210 35 88 316 370 183 52 158 355 1102 91 2708 139 148 275 80 1099 65 173 35 110 279 164 443 AauI BglI EciI AatI NsiI ø1.6 øOL A1 A2 A2 C ø1.1A ø1.1B ø1.3 ø1.5 TR/ SRL BoxA R0.3 0.3 0.4 R0.5 0.5 0.6A/B 0.7 R1 1 R1.1 1.1 1.2 R1.3 1.3 TE 1.4 1.5 1.6 1.7 A0 1762 35 213 270 69 408 2115 357 464 478 464 903 114 73 255 267 402 300 223 172 Ø4c Ø4.7 Ø6.5 Ø4.3 4A/4B/4.1/4.2 4.3 7.7 R4.7 R6.5 4.7 5 5.3 5.5 5.7 5.9 6 6.3 6.5 6.7 7 7.3 4.5 D50L BsiWI D51L PvuI D52L EcoRI D53L SacII D54L BamHI D55L XmaI D56L ApaI D57L HindIII FspI AvrII D50R D51R D52R D53R D54R D55R D56R D57R XbaI D57L HindIII D59L EcoRI D60L BsiWI D61L PvuI D62L EagI D58L PfoI RsrII BssHII AatII AvaI PacI D57R D58R D59R D60R D61R D62R ScaI NdeI PacI NciI AvaI BstBI ApaLI 1611 35 1038 35 464 73 591 2382 3 ø9 ø10 2382 3 62 417 591 2244 3520 437 8 10A Tø 12 9 11 ø13 12 13 14 16 R13 15 D64L HindIII D65L BamHI D66L XmaI D67L ApaI D68L EcoRI D69L BsiWI D70L PstI D71L SalI D63L SacII D63R D64R D65R D66R D67R D68R D69R D70R D71R BglI 35 1662 204 270 53 460 1761 35 150 160 E ø17 øOR 17 18 R18.5 18.5/18.7 19/19.2/19.3 19.5 17.5 SRR/TR

  15. E.colibrator, Polkadorks @ IAP 2004

  16. Struggle, Limited Success, Struggle… Devices? System?? Design & Fabrication Application

  17. Struggle, Success, Predictable Success Tools Registries Measurement Synthesis Applications Systems Parts & Fabrication Design Application

  18. Enabling Biological Engineering • 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

  19. Abstraction Hierarchy Systems Devices Parts

  20. Parts Zif268, Paveltich & Pabo c. 1991

  21. CI LacI Devices CI LacI RBS l cI-857 OLac T

  22. Devices LacI  CI inverter LacI CI

  23. Systems Inverter.1 Inverter.2 Inverter.3

  24. Zif268, Paveltich & Pabo c. 1991 Interfaces Systems Inv.1 Inv.2 Inv.3 LacI  CI inverter LacI CI Devices Parts

  25. A  B inverter A B Zif268, Paveltich & Pabo c. 1991 Parts/Device Interface LacI  CI inverter LacI CI Devices X X Parts

  26. A  B inverter C  D inverter E  F inverter A C E B D F Device/System Interface Inv.1 Inv.2 Inv.3 Systems Devices

  27. E  F inverter A  B inverter C  D inverter A E C D B F Device/System Interface X A  B E  F C  D X X Systems Devices

  28. C  D inverter E  F inverter A  B inverter E C A F D B Device/System Interface A  D A  B E  F C  D X X Systems X Devices

  29. cI LacI cI PoPSin RBS l cI-857 OLac T RBS l cI-857 Ol T PoPSout cI PoPSout PoPSin LacI Device/System Interface

  30. PoPSOUT PoPSIN INVERTER PoPSOUT PoPSIN Polymerase Per Second = PoPS! PoPSOUT PoPSIN T RBS l cI Ol cI

  31. Device/System Interface A B C Systems X A PoPSOUT Devices PoPSIN B PoPSOUT PoPSIN C PoPSOUT PoPSIN

  32. Zif268, Paveltich & Pabo c. 1991 Interfaces Systems PoPS Inv.1 PoPS Inv.2 PoPS Inv.3 PoPS Inv.1 Devices PoPS PoPS Parts

  33. E.colibrator, Polkadorks @ IAP 2004

  34. Device-Level System Diagram

  35. Parts- and Device-Level System Diagram

  36. DNA Layout

  37. System-Level Timing Diagram

  38. Population-Level Simulations

  39. Characterization and Debug Trigger Test Circuit

  40. MIT Registry of Standard Biological Parts http://parts.mit.edu/

  41. 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

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

  43. Hacking the Living World?

  44. Biological Risk: Background Technology Classes Relevant to Biological Risk (current relative capabilities) Risk Manipulation Detection Analysis Response

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

  46. Biological Risk: Future Strategy Technology Classes Relevant to Future Biological Risk (needed capabilities) Risk Manipulation Detection Analysis Response

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

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