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Presentation given to NEB, Ipswich, MA
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• Bio-manufacturing
• Therapeutics
• Better crops
• Bioremediation
• Energy production
1976
• Bio-manufacturing
• Therapeutics
• Better crops
• Bioremediation
• Energy production
2006
Engineering design cycle
Design
ConstructionTesting
Design cycle is slow in biology
Design
ConstructionTesting
(unpredictable)
(slow)(poor technology)
StandardizationRefinement
MeasurementReuse
Standardization
BioBrick® standard parts
EcoRI XbaI SpeI PstIBioBrick part
Knight, 2003
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
A
B
C
D
E
F
G
H
AB
CD
EF
GH
ABCD
EFGH
Why use BioBrick® parts?
To assemble multi-part systems
Vector assembly from parts
Shetty et al., J Biol Eng, 2008
pSB4K5-I52002:BioBrick vector
4 K
**
E X S P1
x 7
Multi-gene pathways
Gene A Gene B Gene C
Refinement
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
LuxCDABELuxILuxR
Device Engineers
Biologists
AHLLuxR GFP (LVA)
luxP(L)
luxP(R)
Weiss & Knight, 2000
R0040 B0034 C0062 B0015 R0062
Canton et al., Nat Biotech, 2008
BBa_F2620 PoPS3OC6HSL
Canton et al., Nat Biotech, 2008
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
Sender
Receiver Output
Quorum-SensingSystem
Sender Receiver
Output
Input
Two-populationcell-cell signalingpathway
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
Measurement
Standard unit: the Ohm
Promoter reference
BBa_J23101tttacagctagctcagtcctaggtattatgctagc
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
Measurement kit instructions
Promoter strength in AU
Promoter strength in SPU
Promoter strength across laboratories
C.V.=0.113MIT, Virginia Tech, Johns Hopkins, Berkeley, LBL, Harvard, Brown
Characterized promoter library
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
Reuse
Cellular Chassis
System
Replication Transcription Translation Degradation
The host cell is a chassis
Cellular Chassis
System
VirtualMachine
A biological virtual machine
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
Decouple system and chassisChassis gene Engineered System
Ribosomes
RNA Polymerases
Ribosomes
RNA PolymerasesChassis gene Engineered System
O-ribosomes
T7 RNAP
a
b
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
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)
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
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)
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
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
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)
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
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
StandardizationRefinement
MeasurementReuse
Make it funMake it safeMake it open
Constructive synthetic biology
iGEM is a proof-of-concept
