Making biology easier to engineer - September 18, 2008

Reshma Shettyrshetty@ginkgobioworks.com

Making biology easier to engineer

• 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

Thank you

team@ginkgobioworks.com

http://ginkgobioworks.com