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

  26. 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)

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

  28. 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

  29. 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

  30. 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

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

  32. PoPSOUT PoPS Source (Any) PoPSOUT PoPSIN Polymerase Per Second = PoPS! PoPSOUT PoPSIN T RBS l cI Ol cI PoPSOUT PoPSIN INVERTER

  33. PoPSIN.1 PoPS  PoPS 2-input NOR PoPSOUT PoPSOUT PoPSIN.1 PoPSIN.2 T RBS l cI Ol CI PoPSIN.2 T RBS l cI PoPS Fan-In

  34. PoPSOUT.1 PoPSIN T RBS l cI Ol CI PoPSOUT.2 T Ol PoPSOUT.1 PoPS  PoPS 2-output NOT PoPSIN PoPSOUT.2 PoPS Fan-Out

  35. PoPSIN.1 PoPS  PoPS 2-input NOR PoPSOUT PoPSIN.2 PoPSOUT.1 PoPS  PoPS 2-output NOT PoPSIN PoPSOUT.2 PoPS-based Composition

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

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

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

  39. E.colibrator, Polkadorks @ IAP 2004

  40. Device-Level System Diagram

  41. Parts- and Device-Level System Diagram

  42. DNA Layout

  43. Population-Level Simulations

  44. Characterization and Debug Trigger Test Circuit

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

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

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