Benefits to Cadence of the MIT MAP
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Design of Self-Replicating Machines Encoding SRM Atrophy Endy, BIOS, June 2004 SRM SRM Physical Adapt Performance & Learn (short) (medium) SRM time Evolve or Persist (long) 1 Consider a Cell Signaling Pathway Cell Yeast Cycle Endy, BIOS, June 2004 2 Consider a Different Cell Budding Yet Another Signaling Pathway Signaling Cell Pathway Yeast Cycle Metabolism Endy, BIOS, June 2004 3 Endy, BIOS, June 2004 4 What does T7 know? Encoding SRMT7 Atrophy Endy, BIOS, June 2004 5 Amber/Essential Amber/Enzyme Deletion F. William Studier, Science 176:403 (1972) Endy, BIOS, June 2004 6 Previous Page Sequence (BNL) Dunn & Studier, J. Mol. Bio. 166:477 (1983) Endy, BIOS, June 2004 7 What do we know? Encoding What We Need To Know SRMT7 What We Know Atrophy Endy, BIOS, June 2004 8 Genome Domestication Encoding What We Make SRMT7-SB Atrophy Endy, BIOS, June 2004 9 NsiI TR/ SRL A0 øOL A1 A2 A2 BoxA Endy, BIOS, June 2004 B R0.30.30.4R0.5 0.5 0.6A/B PciI C 0.7 R1 MfeI 1 SpeI ø1.1A R1.1 ø1.1B ø1.3 1.1 1.2 R1.3 BclI ø1.6 ø1.5 1.3 TE 1.4 1.5 1.61.7 10 NsiI 210 TR/ SRL 63 A0 35 88 316 øOL A1 A2 A2 BoxA Endy, BIOS, June 2004 370 R0.3 0.3 183 52 158 0.4 R0.5 0.5 355 0.6A/B PciI 1102 0.7 MfeI 91 C R1 SpeI 2708 1 139 ø1.1A R1.1 148 ø1.1B 1.1 275 80 ø1.3 1.2 R1.3 1099 65 1.3 TE 173 1.4 BclI 35 ø1.5 110 1.5 35 ø1.6 279 1.6 164 443 1.7 11 SacI 210 TR/ SRL 63 A0 35 88 316 øOL A1 A2 A2 BoxA Endy, BIOS, June 2004 370 R0.3 0.3 183 NheI 52 158 0.4 R0.5 0.5 NsiI 355 0.6A/B PciI 1102 0.7 MfeI 91 C R1 ApaLI 2708 1 139 ø1.1A R1.1 148 ø1.1B 1.1 275 SpeI 80 ø1.3 1.2 R1.3 1099 65 1.3 TE 173 1.4 SapI 35 ø1.5 110 1.5 BclI 35 ø1.6 279 1.6 164 443 1.7 12 D1L BstEII D1R D2L SphI D3L D4L BspDI HindIII D2R D3R 35 88 D5L D6L BssHII SexAI D4R D5R D7L MluI D6R D7R SacI 210 TR/ SRL 63 A0 316 øOL A1 A2 A2 BoxA Endy, BIOS, June 2004 370 R0.3 0.3 183 D8L BsiWI D9L RsrII D8R NheI 52 158 0.4 R0.5 0.5 D9.5L SacII D9R D10L EagI D9.5R NsiI PciI 355 0.6A/B 1102 0.7 D10.5L PacI D11L EcoRI D10R D12L PfoI D10.5R D11R MfeI 91 C R1 D12R D15L ApaI D13R D14R D15R 80 1099 65 ApaLI 2708 1 D13L D14L EcoO1091 XmaI 139 ø1.1A R1.1 148 ø1.1B 1.1 275 D16L NcoI D17L KasI D18L AvrII D16R D17R SpeI ø1.3 1.2 R1.3 1.3 TE 173 1.4 D20L D21L XbaI AgeI D19L AatII D18R D19R D20R 35 279 SapI 35 ø1.5 110 1.5 D21R BclI ø1.6 1.6 164 443 1.7 13 Endy, BIOS, June 2004 14 T7+ Endy, BIOS, June 2004 Rebuild[1-8311]:T7+ 15 T7+ (1) T7+ (2) RB-alpha (1) RB-alpha (2) 60 50 Klett 40 30 20 10 0 -150 -125 -100 -75 -50 -25 0 25 50 time []=minutes Endy, BIOS, June 2004 16 D2L SphI D1L BstEII D1R D3L D4L BspDI HindIII D2R D3R 35 88 D5L D6L BssHII SexAI D4R D5R D7L MluI D6R TR/ SRL 63 A0 370 316 øOL A1 A2 A2 BoxA D9R D10L EagI D9.5R 355 1102 0.6A/B 0.7 D24L D25L D23R EcoRI D24R XmaI D10.5L PacI D11L EcoRI D10R NsiI PciI 158 0.4 R0.5 0.5 D23L D22R AvrII D22L BstEII 52 D9.5L SacII D8R NheI 183 R0.3 0.3 D9L RsrII D7R SacI 210 D8L BsiWI D12L PfoI D10.5R D11R D12R MfeI 91 C 1 D26L D25R BamHI D15L ApaI D16L NcoI D13R D14R D15R 139 ø1.1A R1.1 D27L D26R EagI 148 80 1099 65 ø1.1B ø1.3 1.2 R1.3 D28L D27R SacII D18L AvrII D29L D28R PciI D19R D20R 35 279 D21R SapI 173 1.3 TE D20L D21L XbaI AgeI D19L AatII D18R SpeI 275 1.1 D17L KasI D16R D17R ApaLI 2708 R1 D13L D14L EcoO1091 XmaI ø1.5 1.4 BclI 110 35 1.5 D30L D29R SalI ø1.6 164 443 1.6 1.7 D30R BglII 175 209 1.8 D31L BsiWI D31R XbaI D50L BsiWI 438 2.5 2.8 ø2.5 2 35 Ø4.3 Ø4c 723 464 478 3 75 ø3.8 3.5 D32L D33L D34L D35L D36L D37L D38L D39L D40L D41L D42L D43L D44L EagID32R AscID33R SgrID34R HindIII D35R PfoI D36R NheI SphI NcoI BamHI SacIID41R ApaI XmaI SacI D37R D38R D39R D40R D42R D43R AauI BglI EciI ScaI AvaI SpeI 1762 4A/4B/4.1/4.2 35 213 270 4.3 4.5 D51L D52L D50R PvuI D51R EcoRI DraI BspHI 69 Ø4.7 R4.7 D53L D52R SacII D53R NsiI 408 RsrII 2115 4.7 D54L BamHI XbaI 5 357 464 478 464 5.3 5.5 5.7 5.9 D55L FspI D54R XmaI AseI D56L D55R ApaI BssHII 903 114 AatII 3.8 D45L D46L D47L D48L D49L EcoRI PvuI RsrII PstI D44R XhoI D45R D46R D47R D48R AatI XcaI NsiI 73 Ø6.5 D57R 75 R3.8 6.3 R6.5 6 D57L D56R HindIII EciI 306 255 267 6.5 6.7 D57L HindIII 402 7 BstEII 223 172 7.7 7.3 D59L AvrII D58R EcoRI D58L D57R PfoI AvaI PacI 300 D49R PacI NdeI 1611 35 ø9 8 D60L D61L D59R BsiWI D60R PvuI 14 AvaI 2244 15 Endy, BIOS, June 2004 35 464 ø10 9 D62L D61R EagI NciI 591 1038 10A D64L D63L D62RSacII D63R HindIII ScaI BstBI ApaLI 3520 16 437 ø17 591 Tø 1662 17 KasI BglI 2382 D67L D68L D66R ApaI D67R EcoRI BlpI 204 17.5 270 18 2382 53 460 E 3 12 D69L D68R BsiWI BspDI R18.5 3 12 11 D65L D66L D64R BamHI D65R XmaI FspI 35 73 D70L D69R PstI DraIII AcvI 62 R13 417 ø13 D71L D70R SalI 13 D71R AciI 1761 18.5/18.7 19/19.2/19.3 KpnI 35 øOR 150 19.5 160 SRR/TR 17 Endy, BIOS, June 2004 18 E.colibrator, Polkadorks @ IAP 2004 QuickTime™ and a Video decompressor are needed to see this picture. Endy, BIOS, June 2004 19 Struggle, Limited Success, Struggle… Design & Fabrication Endy, BIOS, June 2004 Devices? System?? Application 20 Struggle, Success, Predictable Success Design Parts & Fabrication Systems Tools Registries Measurement Applications Application Synthesis Endy, BIOS, June 2004 21 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 Endy, BIOS, June 2004 22 Abstraction Hierarchy Systems Devices Parts Endy, BIOS, June 2004 23 Parts Zif268, Paveltich & Pabo c. 1991 Endy, BIOS, June 2004 24 Devices CI LacI OLac RBS l cI-857 T CI LacI Endy, BIOS, June 2004 25 Devices LacI CI LacI CI inverter Endy, BIOS, June 2004 26 Systems Inverter.1 Endy, BIOS, June 2004 Inverter.2 Inverter.3 27 Interfaces Systems Devices Inv.1 Inv.2 Inv.3 LacI LacI CI inverter CI Parts Zif268, Paveltich & Pabo c. 1991 Endy, BIOS, June 2004 28 Parts/Device Interface LacI Devices LacI CI inverter CI Parts Zif268, Paveltich & Pabo c. 1991 Endy, BIOS, June 2004 29 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) Endy, BIOS, June 2004 30 Parts/Device Interface A LacI X Devices LacI A CI B inverter B CI X Parts Zif268, Paveltich & Pabo c. 1991 Endy, BIOS, June 2004 31 Device/System Interface Systems Devices Endy, BIOS, June 2004 Inv.1 Inv.2 Inv.3 A AB inverter B C CD inverter D E EF inverter F 32 Device/System Interface X Systems Devices Endy, BIOS, June 2004 X AB EF X CD A AB inverter B C CD inverter D E EF inverter F 33 Device/System Interface AD Systems X AB EF X CD X Devices Endy, BIOS, June 2004 A AB inverter B C CD inverter D E EF inverter F 34 Device/System Interface cI LacI cI PoPSin OLac RBS l cI-857 T RBS l cI-857 T Ol PoPSout cI PoPSout LacI Endy, BIOS, June 2004 PoPSin 35 Polymerase Per Second = PoPS! PoPSOUT PoPSIN RBS T l cI Ol cI PoPSOUT PoPSOUT PoPSIN PoPS Source (Any) INVERTER PoPSOUT PoPSIN Endy, BIOS, June 2004 36 PoPS Fan-In PoPSOUT PoPSIN.1 RBS l cI T Ol CI PoPSIN.2 RBS l cI T PoPSIN.1 PoPSIN.2 Endy, BIOS, June 2004 PoPSOUT PoPS PoPS 2-input NOR 37 PoPS Fan-Out PoPSOUT.1 PoPSIN RBS l cI T Ol CI PoPSOUT.2 T Ol PoPSOUT.1 PoPSIN PoPS PoPS 2-output NOT Endy, BIOS, June 2004 PoPSOUT.2 38 PoPS-based Composition PoPSOUT.1 PoPSIN PoPS PoPS 2-output NOT PoPSOUT.2 PoPSIN.1 PoPSIN.2 Endy, BIOS, June 2004 PoPSOUT PoPS PoPS 2-input NOR 39 Device/System Interface Systems X Devices Endy, BIOS, June 2004 A AB inverter B C CD inverter D E EF inverter F 40 Device/System Interface A Systems B C X Devices Endy, BIOS, June 2004 PoPSIN A PoPSOUT PoPSIN B PoPSOUT PoPSIN C PoPSOUT 41 Interfaces Systems Devices PoPS PoPS PoPS Inv.1 Inv.2 Inv.3 PoPS PoPS Inv.1 PoPS Parts Zif268, Paveltich & Pabo c. 1991 Endy, BIOS, June 2004 42 E.colibrator, Polkadorks @ IAP 2004 QuickTime™ and a Video decompressor are needed to see this picture. Endy, BIOS, June 2004 43 Device-Level System Diagram Endy, BIOS, June 2004 44 Parts- and Device-Level System Diagram Endy, BIOS, June 2004 45 DNA Layout Endy, BIOS, June 2004 46 Population-Level Simulations QuickTime™ and a Video decompressor are needed to see this picture. Endy, BIOS, June 2004 47 Characterization and Debug Trigger Test Circuit Endy, BIOS, June 2004 48 MIT Registry of Standard Biological Parts http://parts.mit.edu/ Endy, BIOS, June 2004 49 DNA Synthesis 10000000 1000000 100000 Bases of DNA Per Person Day 10000 1000 100 1980 1985 1990 1995 2000 2005 2010 Year Carlson, Pace & Proliferation of Biological Technologies, Biosec. & Bioterror. 1(3):1 (2003) Endy, BIOS, June 2004 50 Hacking the Living World? Endy, BIOS, June 2004 51 Biological Risk: Background Technology Classes Relevant to Biological Risk (current relative capabilities) Manipulation Detection Analysis Risk Response Endy, BIOS, June 2004 52 Biological Risk: Tactics as “Strategy” Anthrax vaccine Maginot Line France, 1940 SARS assay Ciprofloxacin VHF therapy (under construction) Plague vaccine (under construction) Endy, BIOS, June 2004 Smallpox vaccine 53 Biological Risk: Future Strategy Technology Classes Relevant to Future Biological Risk (needed capabilities) Manipulation Detection Risk Analysis Response Endy, BIOS, June 2004 54 Biological Risk: Suite of Solutions Number of Individuals Basic Researcher Garage Bio-Hacker Disgruntled Researcher Bin Laden Genetics, Inc. honorable Endy, BIOS, June 2004 Individual’s Intent dishonorable 55 A Constructive Society Endy, BIOS, June 2004 56 D2L SphI D1L BstEII D1R D3L D4L BspDI HindIII D2R D3R 35 88 D5L D6L BssHII SexAI D4R D5R D7L MluI D6R TR/ SRL 63 A0 370 316 øOL A1 A2 A2 BoxA D9R D10L EagI D9.5R 355 1102 0.6A/B 0.7 D24L D25L D23R EcoRI D24R XmaI D10.5L PacI D11L EcoRI D10R NsiI PciI 158 0.4 R0.5 0.5 D23L D22R AvrII D22L BstEII 52 D9.5L SacII D8R NheI 183 R0.3 0.3 D9L RsrII D7R SacI 210 D8L BsiWI D12L PfoI D10.5R D11R D12R MfeI 91 C 1 D26L D25R BamHI D15L ApaI D16L NcoI D13R D14R D15R 139 ø1.1A R1.1 D27L D26R EagI 148 80 1099 65 ø1.1B ø1.3 1.2 R1.3 D28L D27R SacII D18L AvrII D29L D28R PciI D19R D20R 35 279 D21R SapI 173 1.3 TE D20L D21L XbaI AgeI D19L AatII D18R SpeI 275 1.1 D17L KasI D16R D17R ApaLI 2708 R1 D13L D14L EcoO1091 XmaI ø1.5 1.4 BclI 110 35 1.5 D30L D29R SalI ø1.6 164 443 1.6 1.7 D30R BglII 175 209 1.8 D31L BsiWI D31R XbaI D50L BsiWI 438 2.5 2.8 ø2.5 2 35 Ø4.3 Ø4c 723 464 478 3 75 ø3.8 3.5 D32L D33L D34L D35L D36L D37L D38L D39L D40L D41L D42L D43L D44L EagID32R AscID33R SgrID34R HindIII D35R PfoI D36R NheI SphI NcoI BamHI SacIID41R ApaI XmaI SacI D37R D38R D39R D40R D42R D43R AauI BglI EciI ScaI AvaI SpeI 1762 4A/4B/4.1/4.2 35 213 270 4.3 4.5 D51L D52L D50R PvuI D51R EcoRI DraI BspHI 69 Ø4.7 R4.7 D53L D52R SacII D53R NsiI 408 RsrII 2115 4.7 D54L BamHI XbaI 5 357 464 478 464 5.3 5.5 5.7 5.9 D55L FspI D54R XmaI AseI D56L D55R ApaI BssHII 903 114 AatII 3.8 D45L D46L D47L D48L D49L EcoRI PvuI RsrII PstI D44R XhoI D45R D46R D47R D48R AatI XcaI NsiI 73 Ø6.5 D57R 75 R3.8 6.3 R6.5 6 D57L D56R HindIII EciI 306 255 267 6.5 6.7 D57L HindIII 402 7 BstEII 223 172 7.7 7.3 D59L AvrII D58R EcoRI D58L D57R PfoI AvaI PacI 300 D49R PacI NdeI 1611 35 ø9 8 D60L D61L D59R BsiWI D60R PvuI 14 AvaI 2244 15 Endy, BIOS, June 2004 35 464 ø10 9 D62L D61R EagI NciI 591 1038 10A D64L D63L D62RSacII D63R HindIII ScaI BstBI ApaLI 3520 16 437 ø17 591 Tø 1662 17 KasI BglI 2382 D67L D68L D66R ApaI D67R EcoRI BlpI 204 17.5 270 18 2382 53 460 E 3 12 D69L D68R BsiWI BspDI R18.5 3 12 11 D65L D66L D64R BamHI D65R XmaI FspI 35 73 D70L D69R PstI DraIII AcvI 62 R13 417 ø13 D71L D70R SalI 13 D71R AciI 1761 18.5/18.7 19/19.2/19.3 KpnI 35 øOR 150 19.5 160 SRR/TR 57 Kuroda-Kawaguchi et al., Nature Genetics 29:279 (2001) Endy, BIOS, June 2004 58 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) Endy, BIOS, June 2004 59 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 Endy, BIOS, June 2004 60
