making biology easier to engineer - september 18, 2008
DESCRIPTION
Presentation given to NEB, Ipswich, MATRANSCRIPT
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• Bio-manufacturing
• Therapeutics
• Better crops
• Bioremediation
• Energy production
1976
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• Bio-manufacturing
• Therapeutics
• Better crops
• Bioremediation
• Energy production
2006
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Engineering design cycle
Design
ConstructionTesting
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Design cycle is slow in biology
Design
ConstructionTesting
(unpredictable)
(slow)(poor technology)
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StandardizationRefinement
MeasurementReuse
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Standardization
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BioBrick® standard parts
EcoRI XbaI SpeI PstIBioBrick part
Knight, 2003
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BioBrick® standard assembly
E X S PBioBrick part 1 E X S PBioBrick part 2
Digest with EcoRI and SpeI
Digest with XbaI and PstI
E X SBioBrick part 1 E X S PBioBrick part 2
E X S PBioBrick part 1 BioBrick part 2Mixed
Ligate
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A
B
C
D
E
F
G
H
AB
CD
EF
GH
ABCD
EFGH
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Why use BioBrick® parts?
To assemble multi-part systems
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Vector assembly from parts
Shetty et al., J Biol Eng, 2008
pSB4K5-I52002:BioBrick vector
4 K
**
E X S P1
x 7
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Multi-gene pathways
Gene A Gene B Gene C
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Refinement
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LuxCDABELuxILuxR
1
2
3
4
5
8
Device Engineers
Biologists
AHLLuxR GFP (LVA)
luxP(L)
luxP(R)
R0040 B0034 C0062 B0015 R0062
6BBa_F2620 PoPS3OC6HSL
7System Engineers
Device Engineers
Sender
Receiver Output
Quorum-SensingSystem
Sender Receiver
Output
Input
Two-populationcell-cell signalingpathway
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LuxCDABELuxILuxR
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Device Engineers
Biologists
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AHLLuxR GFP (LVA)
luxP(L)
luxP(R)
Weiss & Knight, 2000
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R0040 B0034 C0062 B0015 R0062
Canton et al., Nat Biotech, 2008
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BBa_F2620 PoPS3OC6HSL
Canton et al., Nat Biotech, 2008
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System Engineers
Device Engineers
Mechanism & FunctionA transcription factor (LuxR) that is active in the presence of a cell-cell signaling molecule (3OC6HSL) is controlled by a regulated operator (PLtetO-1). Device input is 3OC6HSL. Device output is PoPS from a LuxR-regulated operator. If used in a cell containing TetR then a second input such as aTc can be used to produce a Boolean AND function.
BBa_F26203OC6HSL PoPS Receiver
Registry of Standard Biological Parts making life better, one part at a time
License: Public
Conditions (abridged)Output: PoPS measured via BBa_E0240Culture: Supplemented M9, 37ºCPlasmid: pSB3K3Chassis: MG1655*Equipment: PE Victor3 multi-well fluorimeter**Equipment: BD FACScan cytometer
1E01E1
1E21E3
1E4
GFP (arbitrary units)
Doubl
ings
2038
5674
92
High Input (1E -7 M 3OC6HSL)
92
1E01E1
1E21E3
1E4
Doubl
ings
2038
5674
Low Input(0 M 3OC6HSL)
GFP (arbitrary units)
Reliability**
Genetic: >92/>56 culture doublingsPerformance: >92/>56 culture doublings (low/high input during propagation)
Transcriptional Output Demand (low/high input)Nucleotides: 0 / 6xNt nucleotides cell-1 s-1
Polymerases: 0 / 1.5E-1xNt RNAP cell-1
(Nt = downstream transcript length)
Chassis: MC4100, MG1655, and DH5Plasmids: pSB3K3 and pSB1A2Devices: E0240, E0430 and E0434
BBa_F2620 Response Time: <1 minBBa_T9002 Response Time: 6±1 minInputs: 0 M (Low), 1E-07 M (High) 3OC6HSL
Pmax: 6.6 PoPS cell-1 K: 1.5E-09 M 3OC6HSLn: 1.6
Static Performance*
Pout =
Pmax [3OC6HSL]n
Kn + [3OC6HSL]n
0E+00 1E−10 1E−09 1E−08 1E−07 1E−06 1E−05 1E−040
100
200
300
400
500
600
[3OC6HSL] (M)
GFP
synt
hesis
rate
(mol
ecul
es ce
ll−1 s−1
)
Population MeanColony RangeHill Equation
0
1
2
3
4
5
6
7
8
PoPS
cell−1
Part Compatibility (qualitative)
Authors: Barry Canton Ania Labno
Updated: March 2008
Sig
nal
ing
Dev
ices
http
://pa
rts.
mit.
edu/
regi
stry
/inde
x.ph
p/P
art:B
Ba_
F26
20
BBa_F2620
R0040 B0034 C0062 B0015 R0062 PLtetO-1 RBS luxR Term. Plux,R
Component Parts
−10 0 10 20 30 40 500
100
200
300
400
500
600
+ 3OC6HSL
Time (min)
GFP
synt
hesis
rate
(mol
ecul
es ce
ll−1 s−1
)
GFP synthesis rate (Low Input)GFP synthesis rate (High Input)Polynomial Fit (High Input)PoPS (High Input)
0
1
2
3
4
5
6
7
8
PoPS
cell−1
0E+00 1E−10 1E−09 1E−08 1E−07 1E−06 1E−05 1E−040
100
200
300
400
500
600
[AHL] (M)
GFP
synt
hesis
rate
(mol
ecul
es ce
ll−1 s−1
)
C4HSLC6HSL3OC6HSLC7HSLC8HSL3OC8HSLC10HSLC12HSL
0
1
2
3
4
5
6
7
8
PoPS
cell−1
Input Compatibility*
Dynamic Performance*
Canton et al., Nat Biotech, 2008
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Sender
Receiver Output
Quorum-SensingSystem
Sender Receiver
Output
Input
Two-populationcell-cell signalingpathway
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LuxCDABELuxILuxR
1
2
3
4
5
8
Device Engineers
Biologists
AHLLuxR GFP (LVA)
luxP(L)
luxP(R)
R0040 B0034 C0062 B0015 R0062
6BBa_F2620 PoPS3OC6HSL
7System Engineers
Device Engineers
Sender
Receiver Output
Quorum-SensingSystem
Sender Receiver
Output
Input
Two-populationcell-cell signalingpathway
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Measurement
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Standard unit: the Ohm
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Promoter reference
BBa_J23101tttacagctagctcagtcctaggtattatgctagc
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Measurement kit
GFP reporter
pUC AmpR
E X S PE0240
Medium copy BioBrick plasmid
p15A KanR
E X S PP1010
E. coli strain TOP10
Promoter reference standard
p15A KanR
E X S PE0240
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Measurement kit instructions
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Promoter strength in AU
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Promoter strength in SPU
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Promoter strength across laboratories
C.V.=0.113MIT, Virginia Tech, Johns Hopkins, Berkeley, LBL, Harvard, Brown
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Characterized promoter library
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Lessons
•Ad hoc reference standards are useful
•Reference standards materials and instructions should be distributed
•Standards enable comparison of parts
•Standards help to identify sources of variability for part improvement
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Reuse
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Cellular Chassis
System
Replication Transcription Translation Degradation
The host cell is a chassis
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Cellular Chassis
System
VirtualMachine
A biological virtual machine
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Chassis gene Engineered System
Ribosomes
RNA Polymerases
Ribosomes
RNA PolymerasesChassis gene Engineered System
O-ribosomes
T7 RNAP
a
b
System is coupled to the chassis
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Decouple system and chassisChassis gene Engineered System
Ribosomes
RNA Polymerases
Ribosomes
RNA PolymerasesChassis gene Engineered System
O-ribosomes
T7 RNAP
a
b
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Orthogonal transcription and translation (VM1)
O-ribosome generator
mutated rrnB
T7 RNAP
T7 generator
RBSlacUV5
T
Arabinose
IPTG T7 RNAP
O-ribosomesO-ribosome generator
mutated rrnBP
BAD
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GFP expression is specific
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.60
10E+4
20E+4
30E+4
40E+4
Cell Density (OD600)
Fluo
resc
ence
(Rela
tive
Units
)
VM1 + ReporterT7 RNAP + ReporterO-ribosomes + ReporterVM1VM1 (Uninduced)
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O-ribosome generator
mutated rrnB
T7 RNAP
T7 generator
RBSlacUV5
T
Arabinose
IPTG T7 RNAP
O-ribosomesO-ribosome generator
mutated rrnBP
BAD
VM1 consumes RNAP and ribosomes from the cell
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T7 RNAP
O-ribosomes
T7lacM
O-ribosome generator
mutated rrnB
lacI T7 RNAP
T7 autogenerator (BBa_I20257)
O-RBS O-RBST7lacM
T LacI
Auto-regulating T7 RNAP and O-ribosomes (VM2.0)
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0
0.2
0.4
0.6
0.8
1
1.2
VM2.0 T7 RNAPGenerator +
Reporter
O-ribosomeGenerator +
Reporter
VM2.0 +Reporter
Fluo
resc
ence
/OD
(Rel
ativ
e U
nits
)
VM2.0 expresses GFP
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T7 RNAP
O-ribosomes
T7lacM
O-ribosome generator
mutated rrnB
lacI T7 RNAP
T7 autogenerator (BBa_I20257)
O-RBS O-RBST7lacM
T LacI
VM2.0 requires a lacIQ strain
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lacI
T7 autogenerator
o-RBSo-RBST7lacM
T7 RNAP T T
o-ribosomes
T7lacM
o-ribosome generator
mutated rrnB
T7 RNAP
LacI
T7 RNAP
O-ribosomes
T7lacM
O-ribosome generator
mutated rrnB
lacI T7 RNAP
T7 autogenerator (BBa_I20257)
O-RBS O-RBST7lacM
T LacI
Redesigned T7 autogenerator for increased lacI expression
T7 autogenerator (VM2.0)
T7 autogenerator (VM2.2)
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0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.80E+4
0.4E+4
0.8E+4
1.2E+4
1.6E+4
2.0E+4
Cell Density (OD600)
Fluo
resc
ence
(Rela
tive
Units
)
VM2.2 + ReporterT7 RNAP Autogenerator + ReporterO-ribosome Generator + ReporterReporterVM2.2
VM2.2 works in E. coli TOP10
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0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.80E+4
2E+4
4E+4
6E+4
8E+4
10E+4
12E+4
Cell Density (OD600)
Fluo
resc
ence
(Rela
tive
Units
)
VM1.2 + ReporterT7 RNAP + ReporterO-ribosomes + ReporterReporterVM1.2
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.80E+4
2E+4
4E+4
6E+4
8E+4
10E+4
12E+4
Cell Density (OD600)
VM2.2 + ReporterT7 RNAP Autogenerator + ReporterO-ribosome Generator + ReporterReporterVM2.2
lacI
T7 autogenerator
o-RBSo-RBST7lacM
T7 RNAP T T
o-ribosomes
T7lacM
o-ribosome generator
mutated rrnB
T7 RNAP
LacI
O-ribosome generator
mutated rrnB
T7 RNAP
T7 generator
RBSlacUV5
T
Arabinose
IPTG T7 RNAP
O-ribosomesO-ribosome generator
mutated rrnBP
BAD
VM2.2 is less active than VM1
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StandardizationRefinement
MeasurementReuse
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Make it funMake it safeMake it open
Constructive synthetic biology
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iGEM is a proof-of-concept
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