endy, alpha, june 2004 1 atrophy time srm physical performance (short) srm adapt learn (medium) srm...
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1Endy, Alpha, June 2004
Atrophy
timeSRMPhysical
Performance(short)
SRMAdapt
& Learn(medium)
SRMEvolve
or Persist(long)
SRM
Encoding
Design of Self-Replicating Machines
2Endy, Alpha, June 2004
Yeast
Consider a Cell
SignalingPathway
CellCycle
3Endy, Alpha, June 2004
YeastCellCycle
Consider a Different Cell
SignalingPathway
Budding
Metabolism
Yet Another Signaling Pathway
4Endy, Alpha, June 2004
5Endy, Alpha, June 2004
Atrophy
SRMT7
Encoding
What does T7 know?
6Endy, Alpha, June 2004
F. William Studier, Science 176:403 (1972)
Deletion
Amber/Enzyme
Amber/Essential
7Endy, Alpha, June 2004
Previous Page
Sequence (BNL)
Dunn & Studier, J. Mol. Bio. 166:477 (1983)
8Endy, Alpha, June 2004
Atrophy
SRMT7What We
Know
What We Need To Know
Encoding
What do we know?
9Endy, Alpha, June 2004
Atrophy
What We Make
SRMT7-SB
Encoding
Genome Domestication
10Endy, Alpha, June 2004
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
11Endy, Alpha, June 2004
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
12Endy, Alpha, June 2004
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
13Endy, Alpha, June 2004
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
14Endy, Alpha, June 2004
15Endy, Alpha, June 2004
T7+ Rebuild[1-8311]:T7+
16Endy, Alpha, June 2004
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)
17Endy, Alpha, June 2004
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
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
18Endy, Alpha, June 2004
E.colibrator, Polkadorks @ IAP 2004
QuickTime™ and aVideo decompressor
are needed to see this picture.
19Endy, Alpha, June 2004
Application
Struggle, Limited Success, Struggle…
System??Devices?Design &Fabrication
20Endy, Alpha, June 2004
Application
Struggle, Success, Predictable Success
SystemsParts & FabricationDesign Applications
Tools Registries
Synthesis
Measurement
21Endy, Alpha, June 2004
• 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
22Endy, Alpha, June 2004
Abstraction Hierarchy
Devices
Parts
Systems
23Endy, Alpha, June 2004
Parts
Zif268, Paveltich & Pabo c. 1991
24Endy, Alpha, June 2004
Devices
cI-857OLac RBS T
CILacI
LacI
CI
25Endy, Alpha, June 2004
Devices
LacI CI inverter
CILacI
26Endy, Alpha, June 2004
Systems
Inverter.2 Inverter.3Inverter.1
27Endy, Alpha, June 2004
Interfaces
Devices
Parts
SystemsInv.2 Inv.3Inv.1
Zif268, Paveltich & Pabo c. 1991
LacI CI
inverterCILacI
28Endy, Alpha, June 2004
Parts/Device Interface
Devices
Parts
Zif268, Paveltich & Pabo c. 1991
LacI CI
inverterCILacI
X XA B
inverterBA
29Endy, Alpha, June 2004
Device/System Interface
Devices
Systems Inv.2 Inv.3Inv.1
A B inverter
BA
C D inverter
DC
E F inverter
FE
30Endy, Alpha, June 2004
Device/System Interface
Devices
Systems E F C DA B
A B inverter
BA
C D inverter
DC
E F inverter
FE
X XX
31Endy, Alpha, June 2004
Device/System Interface
Devices
Systems E F C DA B
A B inverter
BA
C D inverter
DC
E F inverter
FE
A D
X X
X
32Endy, Alpha, June 2004
Device/System Interface
cI-857OLac RBS T
cILacI
cI-857RBS T
cI
O
PoPSin
PoPSout
LacI
cI PoPSout
PoPSin
33Endy, Alpha, June 2004
cIRBST
O
PoPSOUT
PoPSIN
cI
PoPSOUT
INVERTER
PoPSOUTPoPSIN
PoPSIN
Polymerase Per Second = PoPS!
34Endy, Alpha, June 2004
Device/System Interface
Devices
SystemsB CA
A
B
C
PoPSIN
PoPSIN
PoPSIN
PoPSOUT
PoPSOUT
PoPSOUT
X
35Endy, Alpha, June 2004
Interfaces
Devices
Parts
Systems PoPS Inv.2
PoPS Inv.3
PoPS Inv.1
Zif268, Paveltich & Pabo c. 1991
PoPSInv.1PoPS PoPS
36Endy, Alpha, June 2004
E.colibrator, Polkadorks @ IAP 2004
QuickTime™ and aVideo decompressor
are needed to see this picture.
37Endy, Alpha, June 2004
Device-Level System Diagram
38Endy, Alpha, June 2004
Parts- and Device-Level System Diagram
39Endy, Alpha, June 2004
DNA Layout
40Endy, Alpha, June 2004
System-Level Timing Diagram
41Endy, Alpha, June 2004
Population-Level Simulations
QuickTime™ and aVideo decompressor
are needed to see this picture.
42Endy, Alpha, June 2004
Trigger Test Circuit
Characterization and Debug
43Endy, Alpha, June 2004
MIT Registry of Standard Biological Parts
http://parts.mit.edu/
44Endy, Alpha, June 2004
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
45Endy, Alpha, June 2004
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)
46Endy, Alpha, June 2004
Hacking the Living World?
47Endy, Alpha, June 2004
Risk
Biological Risk: Background
Technology Classes Relevant to Biological Risk
(current relative capabilities)Manipulation
Analysis
Response
Detection
48Endy, Alpha, June 2004
Biological Risk: Tactics as “Strategy”
Maginot LineFrance, 1940
Ciprofloxacin
Smallpox vaccine
Anthrax vaccine
SARS assay
VHF therapy(under construction)
Plague vaccine(under construction)
49Endy, Alpha, June 2004
Biological Risk: Future Strategy
Risk
Technology Classes Relevant to Future Biological Risk
(needed capabilities)
Detection
Analysis
Response
Manipulation
50Endy, Alpha, June 2004
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
51Endy, Alpha, June 2004
A Constructive Society
52Endy, Alpha, June 2004
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
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
53Endy, Alpha, June 2004
Kuroda-Kawaguchi et al., Nature Genetics 29:279 (2001)
54Endy, Alpha, June 2004
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)
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