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

1Endy, Alpha, June 2004

Atrophy

timeSRMPhysical

Performance(short)

SRMAdapt

& Learn(medium)

SRMEvolve

or Persist(long)

SRM

Encoding

Design of Self-Replicating Machines


Yeast

Consider a Cell

SignalingPathway

CellCycle


YeastCellCycle

Consider a Different Cell

SignalingPathway

Budding

Metabolism

Yet Another Signaling Pathway



Atrophy

SRMT7

Encoding

What does T7 know?


F. William Studier, Science 176:403 (1972)

Deletion

Amber/Enzyme

Amber/Essential


Previous Page

Sequence (BNL)

Dunn & Studier, J. Mol. Bio. 166:477 (1983)


Atrophy

SRMT7What We

Know

What We Need To Know

Encoding

What do we know?


Atrophy

What We Make

SRMT7-SB

Encoding

Genome Domestication


A0TR/SRL

øOL A1 A2 R0.3BoxA 0.30.4 0.5R0.5 0.6A/B 0.7 C R1 1 ø1.1A ø1.1BR1.1 1.11.2ø1.3R1.3 1.3 TE 1.4 1.5 1.61.7ø1.5 ø1.6A2

NsiI PciI MfeI SpeI BclI

B


A0

63

TR/SRL

øOL

35

A1

88

A2

316

R0.3BoxA 0.3

370

0.4

183

0.5

158

R0.5

52

0.6A/B

355

0.7

1102 91

CR1 1

2708 139

ø1.1A ø1.1BR1.1 1.1

148

1.2

275 80

ø1.3R1.3 1.3

1099

TE

65

1.4

173

1.5

110

1.6

279

1.7

164

ø1.5

35

ø1.6

35

A2

210 443

NsiI PciI MfeI SpeI BclI


A0

63

TR/SRL

øOL

35

A1

88

A2

316

R0.3BoxA 0.3

370

0.4

183

0.5

158

R0.5

52

0.6A/B

355

0.7

1102 91

CR1 1

2708 139

ø1.1A ø1.1BR1.1 1.1

148

1.2

275 80

ø1.3R1.3 1.3

1099

TE

65

1.4

173

1.5

110

1.6

279

1.7

164

ø1.5

35

ø1.6

35

A2

210 443

NsiI PciI MfeI SpeI BclISacI NheI ApaLI SapI


SacI

D1L BstEII

D1R

A0

D2L SphI

63

TR/SRL

D2R

øOL

D3L BspDI

35

D3R

A1

D4L HindIII

88

D4R

A2

D5L BssHII

316

R0.3BoxA

D6L SexAID5R

0.3

370

D7L MluI

D6R

0.4

183

D8L BsiWI

D7R

0.5

158

R0.5

52

D9L RsrII

D8R

0.6A/B

355

D9.5L SacII

D9R

0.7

1102

D10L EagI

D9.5R

91

CR1

D10.5L PacI

D10R

1

2708

D11L EcoRI

D10.5R

139

ø1.1A ø1.1BR1.1

D12L PfoI

D11R

1.1

148

D13L EcoO1091

D12R

1.2

275

D14L XmaI

D13R

80

ø1.3R1.3

D15L ApaI

D14R

1.3

1099

D16L NcoI

D15R

TE

65

D17L KasI

D16R

1.4

173

D19L AatII

D18R

1.5

110

D21L AgeI

D20R

1.6

279

D21R

1.7

164

D17R

ø1.5

D18L AvrII

35

D19R

ø1.6

D20L XbaI

35

A2

210 443

NheI NsiI PciI MfeI ApaLI SpeI SapI BclI



T7+ Rebuild[1-8311]:T7+


0

10

20

30

40

50

60

-150 -125 -100 -75 -50 -25 0 25 50

time []=minutes

Klett

T7+ (1) T7+ (2) RB-alpha (1) RB-alpha (2)


SacI

D1L BstEII

D1R

A0

D2L SphI

63

TR/SRL

D2R

øOL

D3L BspDI

35

D3R

A1

D4L HindIII

88

D4R

A2

D5L BssHII

316

R0.3BoxA

D6L SexAID5R

0.3

370

D7L MluI

D6R

0.4

183

D8L BsiWI

D7R

0.5

158

R0.5

52

D9L RsrII

D8R

0.6A/B

355

D9.5L SacII

D9R

0.7

1102

D10L EagI

D9.5R

91

CR1

D10.5L PacI

D10R

1

2708

D11L EcoRI

D10.5R

139

ø1.1A ø1.1BR1.1

D12L PfoI

D11R

1.1

148

D13L EcoO1091

D12R

1.2

275

D14L XmaI

D13R

80

ø1.3R1.3

D15L ApaI

D14R

1.3

1099

D16L NcoI

D15R

TE

65

D17L KasI

D16R

1.4

173

D19L AatII

D18R

1.5

110

D21L AgeI

D20R

1.6

279

D21R

1.7

164

D17R

ø1.5

D18L AvrII

35

D19R

ø1.6

D20L XbaI

35

A2

210 443


D23L AvrII

D24L EcoRI

D26L BamHI

D27L EagI

D28L SacII

D30L SalI

D22L BstEII D22R D23R D24R D26R D27R D28R D30R

ø2.5 32.8 ø3.83.5 3.8R3.8

D25L XmaI D25R

2.5

D29L PciI D29R

1.8 2

BglII

175 209 35 723 438 464 478 75 306 75

D32L EagI

D33L AscI

D35L HindIII

D36L PfoI

D37L NheI

D43L XmaI

D31L BsiWI D31R D32R D33R D35R D36R D40R

4.7 5.7

D34L SgrID34R

4.5

D41L SacII

D42R

4A/4B/4.1/4.2 4.3

1762 35 213 270 69 408 478 903 73

Ø4c Ø4.3 Ø4.7R4.7

D38L SphI

D37R

5

2115

D39L NcoI

D38R

5.3

357

D40L BamHI

D39R

5.5

464

D42L ApaID41R

5.9

464

6

D44L SacI

D43R

114

6.3 Ø6.5R6.5

D45L XhoID44R

D46L EcoRI

D45R

255

6.5

D47L PvuI

D46R

267

6.7

D48L RsrII

D47R

402

7

D49L PstI

D48R

300

7.3

D49R

7.7

223 172

BstEIIXbaI AauI BglI EciI ScaIAvaI SpeI AatI XcaI NsiI

FspI AvrIID50L BsiWI

D51L PvuI

D53L SacII

D54L BamHI

D55L XmaI

D57L HindIIID50R D51R D53R D54R D55R D57R

ø9 10A Tø 12

D52L EcoRI D52R

9

D56L ApaI D56R

8

PacI

1611 35 1038 35 464 73 591 2382 3

ø10 11

DraI RsrIIXbaI

BssHII AatII AvaIBspHI EciINsiI

NdeI

AseI

D57L HindIII

D59L EcoRI

D58L PfoID57R D58R

12 13

PacI

2382 62 4173

ø13R13

D60L BsiWI

D62L EagID59R D60R D62R

16

D61L PvuI D61R

15

ApaLI

591 2244 3520 437

14

NciIScaI

BstBIAvaI

D64L HindIII

D65L BamHI

D67L ApaI

D68L EcoRI

D69L BsiWI

D71L SalI

D63L SacII D63R D64R D65R D67R D68R D69R D71R

ø17 18.5/18.718 E 19/19.2/19.3 19.5R18.5

D66L XmaI D66R

17.5

D70L PstI D70R

17

35 1662 204 270 53 460 1761 35 150

øOR SRR/TR

160

KasI BspDIFspIBglI

BlpI DraIII AcvI AciI KpnI


E.colibrator, Polkadorks @ IAP 2004

QuickTime™ and aVideo decompressor

are needed to see this picture.


Application

Struggle, Limited Success, Struggle…

System??Devices?Design &Fabrication


Application

Struggle, Success, Predictable Success

SystemsParts & FabricationDesign Applications

Tools Registries

Synthesis

Measurement


• 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


Abstraction Hierarchy

Devices

Parts

Systems


Parts

Zif268, Paveltich & Pabo c. 1991


Devices

cI-857OLac RBS T

CILacI

LacI

CI


Devices

LacI CI inverter

CILacI


Systems

Inverter.2 Inverter.3Inverter.1


Interfaces

Devices

Parts

SystemsInv.2 Inv.3Inv.1


LacI CI

inverterCILacI


Parts/Device Interface

Devices

Parts


LacI CI

inverterCILacI

X XA B

inverterBA


Device/System Interface

Devices

Systems Inv.2 Inv.3Inv.1

A B inverter

BA

C D inverter

DC

E F inverter

FE



Devices

Systems E F C DA B

A B inverter

BA

C D inverter

DC

E F inverter

FE

X XX



Devices

Systems E F C DA B

A B inverter

BA

C D inverter

DC

E F inverter

FE

A D

X X

X



cI-857OLac RBS T

cILacI

cI-857RBS T

cI

O

PoPSin

PoPSout

LacI

cI PoPSout

PoPSin


cIRBST

O

PoPSOUT

PoPSIN

cI

PoPSOUT

INVERTER

PoPSOUTPoPSIN

PoPSIN

Polymerase Per Second = PoPS!



Devices

SystemsB CA

A

B

C

PoPSIN

PoPSIN

PoPSIN

PoPSOUT

PoPSOUT

PoPSOUT

X


Interfaces

Devices

Parts

Systems PoPS Inv.2

PoPS Inv.3

PoPS Inv.1


PoPSInv.1PoPS PoPS


E.colibrator, Polkadorks @ IAP 2004




Device-Level System Diagram


Parts- and Device-Level System Diagram


DNA Layout


System-Level Timing Diagram


Population-Level Simulations




Trigger Test Circuit

Characterization and Debug


MIT Registry of Standard Biological Parts

http://parts.mit.edu/


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


DNA Synthesis

100

1000

10000

100000

1000000

10000000

1980 1985 1990 1995 2000 2005 2010Year

Bases of DNAPer Person Day

Carlson, Pace & Proliferation of Biological Technologies, Biosec. & Bioterror. 1(3):1 (2003)


Hacking the Living World?


Risk

Biological Risk: Background

Technology Classes Relevant to Biological Risk

(current relative capabilities)Manipulation

Analysis

Response

Detection


Biological Risk: Tactics as “Strategy”

Maginot LineFrance, 1940

Ciprofloxacin

Smallpox vaccine

Anthrax vaccine

SARS assay

VHF therapy(under construction)

Plague vaccine(under construction)


Biological Risk: Future Strategy

Risk

Technology Classes Relevant to Future Biological Risk

(needed capabilities)

Detection

Analysis

Response

Manipulation


Biological Risk: Suite of Solutions

Num

ber

of

Indi

vidu

als

Individual’s Intenthonorable dishonorable

Bin Laden Genetics, Inc.

DisgruntledResearcher

Garage Bio-

Hacker

BasicResearch

er


A Constructive Society


SacI

D1L BstEII

D1R

A0

D2L SphI

63

TR/SRL

D2R

øOL

D3L BspDI

35

D3R

A1

D4L HindIII

88

D4R

A2

D5L BssHII

316

R0.3BoxA

D6L SexAID5R

0.3

370

D7L MluI

D6R

0.4

183

D8L BsiWI

D7R

0.5

158

R0.5

52

D9L RsrII

D8R

0.6A/B

355

D9.5L SacII

D9R

0.7

1102

D10L EagI

D9.5R

91

CR1

D10.5L PacI

D10R

1

2708

D11L EcoRI

D10.5R

139

ø1.1A ø1.1BR1.1

D12L PfoI

D11R

1.1

148

D13L EcoO1091

D12R

1.2

275

D14L XmaI

D13R

80

ø1.3R1.3

D15L ApaI

D14R

1.3

1099

D16L NcoI

D15R

TE

65

D17L KasI

D16R

1.4

173

D19L AatII

D18R

1.5

110

D21L AgeI

D20R

1.6

279

D21R

1.7

164

D17R

ø1.5

D18L AvrII

35

D19R

ø1.6

D20L XbaI

35

A2

210 443


D23L AvrII

D24L EcoRI

D26L BamHI

D27L EagI

D28L SacII

D30L SalI

D22L BstEII D22R D23R D24R D26R D27R D28R D30R

ø2.5 32.8 ø3.83.5 3.8R3.8

D25L XmaI D25R

2.5

D29L PciI D29R

1.8 2

BglII

175 209 35 723 438 464 478 75 306 75

D32L EagI

D33L AscI

D35L HindIII

D36L PfoI

D37L NheI

D43L XmaI

D31L BsiWI D31R D32R D33R D35R D36R D40R

4.7 5.7

D34L SgrID34R

4.5

D41L SacII

D42R

4A/4B/4.1/4.2 4.3

1762 35 213 270 69 408 478 903 73

Ø4c Ø4.3 Ø4.7R4.7

D38L SphI

D37R

5

2115

D39L NcoI

D38R

5.3

357

D40L BamHI

D39R

5.5

464

D42L ApaID41R

5.9

464

6

D44L SacI

D43R

114

6.3 Ø6.5R6.5

D45L XhoID44R

D46L EcoRI

D45R

255

6.5

D47L PvuI

D46R

267

6.7

D48L RsrII

D47R

402

7

D49L PstI

D48R

300

7.3

D49R

7.7

223 172

BstEIIXbaI AauI BglI EciI ScaIAvaI SpeI AatI XcaI NsiI

FspI AvrIID50L BsiWI

D51L PvuI

D53L SacII

D54L BamHI

D55L XmaI

D57L HindIIID50R D51R D53R D54R D55R D57R

ø9 10A Tø 12

D52L EcoRI D52R

9

D56L ApaI D56R

8

PacI

1611 35 1038 35 464 73 591 2382 3

ø10 11

DraI RsrIIXbaI

BssHII AatII AvaIBspHI EciINsiI

NdeI

AseI

D57L HindIII

D59L EcoRI

D58L PfoID57R D58R

12 13

PacI

2382 62 4173

ø13R13

D60L BsiWI

D62L EagID59R D60R D62R

16

D61L PvuI D61R

15

ApaLI

591 2244 3520 437

14

NciIScaI

BstBIAvaI

D64L HindIII

D65L BamHI

D67L ApaI

D68L EcoRI

D69L BsiWI

D71L SalI

D63L SacII D63R D64R D65R D67R D68R D69R D71R

ø17 18.5/18.718 E 19/19.2/19.3 19.5R18.5

D66L XmaI D66R

17.5

D70L PstI D70R

17

35 1662 204 270 53 460 1761 35 150

øOR SRR/TR

160

KasI BspDIFspIBglI

BlpI DraIII AcvI AciI KpnI


Kuroda-Kawaguchi et al., Nature Genetics 29:279 (2001)


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)

Documents

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