Presentation given to NEB, Ipswich, MA

1. Making biology easier to
engineer
Reshma Shetty
rshetty@ginkgobioworks.com

2. 1976
• Bio-manufacturing
• Therapeutics
• Better crops
• Bioremediation
• Energy production

3. 2006
• Bio-manufacturing
• Therapeutics
• Better crops
• Bioremediation
• Energy production

5. Engineering design cycle
Design
Testing Construction

6. Design cycle is slow in biology
Design
(unpredictable)
Testing Construction
(poor technology) (slow)

7. Standardization
Refinement
Measurement
Reuse

8. Standardization

9. BioBrick® standard parts
EcoRI XbaI BioBrick part SpeI PstI
Knight, 2003

10. BioBrick® standard assembly
E X BioBrick part 1 S P E X BioBrick part 2 S P
Digest with Digest with
EcoRI and SpeI XbaI and PstI
E X BioBrick part 1 S E X BioBrick part 2 S P
Ligate
E X BioBrick part 1 Mixed BioBrick part 2 S P

11. A
AB
B
ABCD
C
CD
D
E
EF
F
EFGH
G
GH
H

12. Why use BioBrick® parts?
To assemble multi-part systems

13. Vector assembly from parts
E X 1 S P
* *
pSB4K5-I52002:
BioBrick vector
4 K
x7
Shetty et al., J Biol Eng, 2008

14. Multi-gene pathways
Gene A Gene B Gene C

15. Refinement

17. Two-population
cell-cell signaling Output
pathway Quorum-
Sender Sensing
System
Sender Receiver 8
Receiver Output
Input
System Engineers
7
Device Engineers
BBa_F2620 PoPS 6
3OC6HSL
R0040 B0034 C0062 B0015 R0062
5
AHL
luxP(R)
LuxR GFP (LVA) 4
luxP(L)
Device Engineers
3
Biologists
LuxR LuxI LuxCDABE
2
1

20. LuxR LuxI LuxCDABE

21. Device
Engineers
Biologists

22. AHL
luxP(R)
LuxR GFP (LVA)
luxP(L)
Weiss & Knight, 2000

23. R0040 B0034 C0062 B0015 R0062
Canton et al., Nat Biotech, 2008

24. BBa_F2620 PoPS
3OC6HSL
Canton et al., Nat Biotech, 2008

25. BBa_F2620 BBa_F2620
3OC6HSL PoPS Receiver
Mechanism & Function Component Parts
A 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 R0040 B0034 C0062 B0015 R0062
used in a cell containing TetR then a second input such as PLtetO-1 RBS luxR Term. Plux,R
aTc can be used to produce a Boolean AND function.
Static Performance* Dynamic Performance*
http://parts.mit.edu/registry/index.php/Part:BBa_F2620
600 8 600 8
GFP synthesis rate (molecules cell−1 s−1)
GFP synthesis rate (molecules cell−1 s−1)
Population Mean
Colony Range 7 + 3OC6HSL 7
500 Hill Equation 500
6 6
400 400
5 5
PoPS cell−1
PoPS cell−1
System
300 4 300 4
3 3
200 200 GFP synthesis rate (Low Input)
2 GFP synthesis rate (High Input) 2
100 100 Polynomial Fit (High Input)
1 PoPS (High Input) 1
Engineers
0 0 0 0
0E+00 1E−10 1E−09 1E−08 1E−07 1E−06 1E−05 1E−04 −10 0 10 20 30 40 50
[3OC6HSL] (M) Time (min)
Pmax [3OC6HSL]n Pmax: 6.6 PoPS cell-1 BBa_F2620 Response Time: <1 min
Pout = K: 1.5E-09 M 3OC6HSL BBa_T9002 Response Time: 6±1 min
K n + [3OC6HSL]n n: 1.6 Inputs: 0 M (Low), 1E-07 M (High) 3OC6HSL
Input Compatibility* Reliability**
Device
600 8
C4HSL
GFP synthesis rate (molecules cell−1 s−1)
C6HSL 7
500
3OC6HSL
6
C7HSL 92
400
5 74
C8HSL
gs
PoPS cell−1
56
lin
3OC8HSL
Engineers
300 4 38 92
ub
1E0
C10HSL
Signaling Devices
GFP 1E1
Do
20
(arb1E2 1E3 1E4 74
gs
C12HSL 3 itrar
200 y un 56
lin
its)
ub
2 38
Low Input GFP1E0 1E1
Do
(0 M 3OC6HSL) (arb 1E2 1E3 20
100 itrar
1 y un 1E4
its)
High Input
0 0 (1E -7 M 3OC6HSL)
0E+00 1E−10 1E−09 1E−08 1E−07 1E−06 1E−05 1E−04
[AHL] (M) Genetic: >92/>56 culture doublings
Part Compatibility (qualitative) Performance: >92/>56 culture doublings
(low/high input during propagation)
Chassis: MC4100, MG1655, and DH5
Plasmids: pSB3K3 and pSB1A2
Conditions (abridged)
Devices: E0240, E0430 and E0434
Output: PoPS measured via BBa_E0240
Culture: Supplemented M9, 37ºC
Transcriptional Output Demand (low/high input) Plasmid: pSB3K3
Nucleotides: 0 / 6xNt nucleotides cell-1 s-1 Chassis: MG1655
Polymerases: 0 / 1.5E-1xNt RNAP cell-1 *Equipment: PE Victor3 multi-well fluorimeter
(Nt = downstream transcript length) **Equipment: BD FACScan cytometer
Authors: Barry Canton
Ania Labno Registry of Standard Biological Parts License: Public
Updated: March 2008 making life better, one part at a time
Canton et al., Nat Biotech, 2008

26. Two-population
cell-cell signaling Output
pathway Quorum-
Sender Sensing
System
Sender Receiver
Receiver Output
Input

27. Two-population
cell-cell signaling Output
pathway Quorum-
Sender Sensing
System
Sender Receiver 8
Receiver Output
Input
System Engineers
7
Device Engineers
BBa_F2620 PoPS 6
3OC6HSL
R0040 B0034 C0062 B0015 R0062
5
AHL
luxP(R)
LuxR GFP (LVA) 4
luxP(L)
Device Engineers
3
Biologists
LuxR LuxI LuxCDABE
2
1

28. Measurement

30. Standard unit: the Ohm

31. Promoter reference
BBa_J23101
tttacagctagctcagtcctaggtattatgctagc

32. Measurement kit
E X E0240 S P
E. coli strain
GFP reporter
TOP10
pUC AmpR
E X P1010 S P E X E0240 S P
Medium copy Promoter reference
BioBrick plasmid standard
p15A KanR p15A KanR

33. Measurement kit instructions

34. Promoter strength in AU

35. Promoter strength in SPU

36. Promoter strength
across laboratories
MIT, Virginia Tech, Johns Hopkins, Berkeley, LBL, Harvard, Brown C.V.=0.113

37. Characterized promoter library

38. 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

39. Reuse

40. The host cell is a chassis
System
Replication Transcription Translation Degradation
Cellular Chassis

41. A biological virtual machine
System
Virtual
Machine
Cellular Chassis

42. System is coupled to the
chassis
a
Ribosomes
RNA Polymerases
Chassis gene Engineered System
b
Ribosomes O-ribosomes

43. Decouple system and chassis
RNA Polymerases
Chassis gene Engineered System
b
Ribosomes O-ribosomes
T7 RNAP
RNA Polymerases
Chassis gene Engineered System

44. Orthogonal transcription and
translation (VM1)
O-ribosome generator
O-ribosome generator
O-ribosomes
mutated rrnB rrnB
mutated
PBAD
Arabinose
T7 generator
RBS T7 RNAP T
lacUV5
IPTG T7 RNAP

45. GFP expression is specific
40E+4
VM1 + Reporter
T7 RNAP + Reporter
O-ribosomes + Reporter
Fluorescence (Relative Units)
VM1
30E+4 VM1 (Uninduced)
20E+4
10E+4
0
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
Cell Density (OD600)

46. VM1 consumes RNAP and
ribosomes from the cell
O-ribosome generator
O-ribosome generator
O-ribosomes
mutated rrnB rrnB
mutated
PBAD
Arabinose
T7 generator
RBS T7 RNAP T
lacUV5
IPTG T7 RNAP

47. Auto-regulating T7 RNAP and
O-ribosomes (VM2.0)
O-ribosome generator
O-ribosomes
mutated rrnB
T7lacM
T7 autogenerator (BBa_I20257)
O-RBS lacI O-RBS T7 RNAP T LacI
T7lacM
T7 RNAP

48. VM2.0 expresses GFP
1.2
Fluorescence/OD (Relative Units)
1
0.8
0.6
0.4
0.2
0
VM2.0 T7 RNAP O-ribosome VM2.0 +
Generator + Generator + Reporter
Reporter Reporter

49. VM2.0 requires a lacIQ strain
O-ribosome generator
O-ribosomes
mutated rrnB
T7lacM
T7 autogenerator (BBa_I20257)
O-RBS lacI O-RBS T7 RNAP T LacI
T7lacM
T7 RNAP

50. O-ribo
Redesigned T7 autogenerator
mutated rrnB
T7lacM
for increased lacI expression
o-ribosome autogenerator (BBa_I20257)
T7 generator
o-ribo
T7 autogenerator mutated rrnBO-RBS
O-RBS lacI T7 RNAP T
T7lacM
(VM2.0) T7lacM
T7
T7 autogenerator
T7 autogenerator o-RBS T7 RNAP T o-RBS lacI T
(VM2.2) T7lacM
T7

51. VM2.2 works in E. coli TOP10
2.0E+4
VM2.2 + Reporter
T7 RNAP Autogenerator + Reporter
O-ribosome Generator + Reporter
Fluorescence (Relative Units)
1.6E+4 Reporter
VM2.2
1.2E+4
0.8E+4
0.4E+4
0E+4
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
Cell Density (OD600)

52. VM2.2 is less active than VM1
12E+4 12E+4
VM1.2 + Reporter VM2.2 + Reporter
T7 RNAP + Reporter T7 RNAP Autogenerator + Reporter
10E+4 O-ribosomes + Reporter 10E+4 O-ribosome Generator + Reporter
Fluorescence (Relative Units)
Reporter Reporter
VM1.2
VM2.2
8E+4 8E+4
6E+4 6E+4
4E+4 4E+4
2E+4 2E+4
0E+4 0E+4
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8
Cell Density (OD600) Cell Density (OD600)
O-ribosome generator o-ribosome generator
O-ribosome generator
O-ribosomes o-ribosomes
mutated rrnB rrnB
mutated mutated rrnB
PBAD T7lacM
Arabinose
T7 autogenerator
T7 generator
LacI
o-RBS T7 RNAP T o-RBS lacI T
RBS T7 RNAP T T7lacM
lacUV5
IPTG T7 RNAP T7 RNAP

53. Standardization
Refinement
Measurement
Reuse

54. Constructive synthetic biology
Make it fun
Make it safe
Make it open

56. iGEM is a proof-of-concept

58. Thank you
team@ginkgobioworks.com
http://ginkgobioworks.com