Technological Advances In Synthetic Biology: Subhayu Basu

Technological advances in synthetic biology Subhayu Basu

Overview • Engineering synthetic multicellular systems • Technology for high diversity and high fidelity library construction • Thoughts on next generation sequencing platforms

Synthetic Biology

 Program cell populations to perform various tasks reliably Regulation: digital & analog components and networks Coordination: cell-cell communication Interface: external and internal sensors, effectors

Bottom-up, Modular Design tissues, cultures

networks

cells computers

pathways modules

gates

CAK

P P

Cdc2 P

P

physical layer

biochemical reactions

CDS

proteins, genes...

Circuit Building Block – Inverter 0

1

input protein (repressor)

P

output protein

CDS

Transcription / Translation

1

0

input protein (repressor)

P

output protein

CDS

Goal: Build complex circuits out of simple, well-characterized devices

Transcription / Translation

Interacting with Cells – Implies Gate 0 0

1

P

CDS

0 1

CDS

0

P

1

P

1 0

CDS

1 1

1

P

CDS

“Rational” Circuit Design -IPTG

+IPTG

Inverter circuit LacI

lacI

EYFP

plac

“ideal”

cI

λ

eyfp P(R)

CI

LacI

lacI

plac

cI

λ

eyfp P(R)

“Rational” Circuit Design -IPTG

+IPTG

Inverter circuit LacI

lacI

EYFP

plac

cI

λ

eyfp P(R)

ure s a me

sim ula

te

CI

LacI

lacI

plac

RB S 2nd1:stmutate : modifyoperator RBS

cI

λ

eyfp P(R)

mutate

Directed Evolution -IPTG

+IPTG

Inverter circuit

lacI

plac

cI

λP(R)

CI

LacI

EYFP

lacI

eyfp

initial sequence

plac

cI

λP(R)

eyfp

mutations selection / screening

select clones for the next generation

Fitness

LacI

Mutant #

Yokobayashi, Weiss, Arnold, PNAS (200

Circuits for Programmed Sender & Receiver Sender cells

Receiver cells

3OC6HSL

3OC6HSL

+ P(tet)

tetR

aTc

P(Ltet-O1)

luxI

Lux P(L)

Sender cells

0 aTc

tetR

luxI 3OC6HSL

luxR

Lux P(R)

GFP(LVA)

Receiver cells 0

LuxR

GFP

3OC6HSL

aTc

Weiss & Knight, DNA6 (2000

Synthetic Multicellular System: Pulse Generator aTc

AHL

Sender

P(tet)

tetR

P(Ltet-O1)

luxI

AHL

luxR

Lux PL

Pulse Generator

Lux PR

cI(LVA)

Lux PR-cI-OR

GFP(LVA)

Modified Genetic Circuit LuxR

0 aTc

tetR

0

luxI 3OC6HSL

cI

AHL

aTc

GFP

Modified Logic Circuit Feed-forward motif creates a race condition (static “0”

Basu, et al., PNAS (200

Synthetic Multicellular System: Pulse Generator AHL

P(tet)

tetR

P(Ltet-O1)

luxI

Sender cell P(tet)

LuxI/LuxR from Vibrio fischeri TetR from transposon Tn10 CI from bacteriophage λ Lux P(R) / CI OR1 hybrid promoter

luxR

Lux P(R)

cI

Lux P(R)*

cI

Pulse Generator

Basu, et al., PNAS (200

Synthetic Multicellular System: Pulse Generator AHL

P(tet)

tetR

P(Ltet-O1)

luxI

Sender cell P(tet)

LuxI/LuxR from Vibrio fischeri TetR from transposon Tn10 CI from bacteriophage λ Lux P(R) / CI OR1 hybrid promoter

luxR

Lux P(R)

cI

Lux P(R)*

GFP

Pulse Generator

Basu, et al., PNAS (200

Engineering Pulse Characteristics

Experimental pulses

Model of pulse gain

• Qualitative model predictions correlate with experiments • Carefully choose genetic parameters • “Sweet spot” when altering RBS and cI repression efficiencies

receivers

senders

Gradual Increase in Signal Level experiments

• Much more common in Nature!

AHL Input

50

AHL (nM)

40

30

Infinite

20

simulations

0.94 nM/min 0.47 nM/min 10

0.31 nM/min 0.24 nM/min

0 0

50

100

150

200

Time (min)

Max amplitude depends on rate  Cells “computing first derivative”!

Spatiotemporal Behavior

Experiments

Model

Models and experiments show that receiver cells react to signal only when they are close to the senders (not possible without “signal processing”)

Natural Pattern Formation

• Robust global behavior from unreliable parts • Repeated network motifs. Same molecules used by different species, different stages of development.

Programmed Pattern Formation LuxR

AHL

LacIM1

CI LacI

LuxI

GFP

Sender

Band detector S0

Signal?

strong CI ON

LacIM1 ON

LacI OFF

medium CI ON

LacIM1 OFF

LacI OFF

weak CI OFF

LacIM1 OFF

LacI ON

GFP OFF

GFP ON

GFP OFF

End

End

End

Basu, et al., Nature (2005

Programmed Pattern Formation LuxR

AHL

LuxR LacIM1

CI LacI

LuxI

LacI GFP

Sender

LacIM1

CI

GFP

Band detector S0

Signal?

strong CI ON

LacIM1 ON

LacI OFF

medium CI ON

LacIM1 OFF

LacI OFF

weak CI OFF

LacIM1 OFF

LacI ON

GFP OFF

GFP ON

GFP OFF

End

End

End

Basu, et al., Nature (2005

Programmed Pattern Formation LuxR

AHL

LuxR LacIM1

CI LacI

LuxI

Sender

LacIM1

CI LacI

GFP

LuxR LacIM1

CI LacI

GFP

GFP

Band detector S0

Signal?

strong CI ON

LacIM1 ON

LacI OFF

medium CI ON

LacIM1 OFF

LacI OFF

weak CI OFF

LacIM1 OFF

LacI ON

GFP OFF

GFP ON

GFP OFF

End

End

End

Basu, et al., Nature (2005

Programmed Pattern Formation LuxR

AHL

LuxR LacIM1

CI LacI

LuxI

Sender

LacIM1

CI LacI

GFP

LuxR LacIM1

CI LacI

GFP

GFP

Band detector S0

Signal?

strong CI ON

LacIM1 ON

LacI OFF

medium CI ON

LacIM1 OFF

LacI OFF

weak CI OFF

LacIM1 OFF

LacI ON

GFP OFF

GFP ON

GFP OFF

End

End

End

Basu, et al., Nature (2005

Programmed Pattern Formation LuxR

AHL

LuxR LacIM1

CI LacI

LuxI

Sender

LacIM1

CI LacI

GFP

LuxR LacIM1

CI LacI

GFP

GFP

Band detector S0

Signal?

strong CI ON

LacIM1 ON

LacI OFF

medium CI ON

LacIM1 OFF

LacI OFF

weak CI OFF

LacIM1 OFF

LacI ON

GFP OFF

GFP ON

GFP OFF

End

End

End

Basu, et al., Nature (2005

40

Band Detect Modules L A

AHL

inverter

(1)

L+L* C (2)

(4)

GFP

L* C

20 10 0 -3 10

(3)

-1

10

0

10

1

10

20 10 0 -3 10 3

CI (uM)

2

BD1 – Hypersensitive LuxR BD2 – Wildtype LuxR BD2’ – Reduced plasmid copy number for LuxR BD3 – Reduced plasmid copy number for LuxR, LacIM1 ,

-2

10

30

LacI (uM)

A

GFP

+

low detect

30 GFP (uM)

high detect

BD1 BD2 BD2' BD3

BD1 BD2 BD2' BD3 -2

-1

10

10

0

10

1

10

BD1 BD2 BD2' BD3

1 0 -3 10

-2

10

-1

10

AHL (uM)

0

10

1

10

Experimental Dosage Response 100 HD1 HD2 HD3

75

75

50

50

25

25

0 10 -4 10 -3 10 -2 10 -1 10 0 10 1

AHL (uM)

0 10 -4 10 -3 10 -2 10 -1 10 0 10 1 AHL (uM)

Cam(r) lacIM1

cI T0

T0 pHTSUB-104

(L VA )

LuxP(R) LuxP(L) LuxR

Plac

T1

Kan(r)

ColE1 G FP

PLux(R)

A p15

HD1 Mutation

pLD T1

lacI

P(R -O1 2)

HD3 Mutation

BD1 BD2 BD3

λ

Fluorescence (A.U.)

100

Bullseye with BD2-Red / BD3GFP

Bullseye with BD2-Red / BD3GFP

Green

5mm

Red

The behavior of the system depends on • the genetic program • the initial conditions (i.e. initial ‘state’) • the environment •…

Thank you

Experimental Spatiotemporal Behavior

senders

receivers

Spatiotemporal Simulations – Shift 40

40

Time (hrs)

-0.2

10

30

-0.4

10

20

0

20

-0.8

10

10 0

2

4

6

8 10

Distance (mm)

10

30

-0.6

10

10

-0.2

0

-0.4

10

-0.6

10

-0.8

10 0

2

4

6

8 10

Distance (mm)

• Analysis of the effect of kinetic parameters on positional shift through simulations

Regression Analysis

LacI

• Generated sets with random kinetic rates • Selected for band-detect behavior (~30%) • Regression analysis to find correlations

40 20 0 0.01

0.1 LacI decay, γL (min-1 )

CI

AHL

LacI

LacI GFP

R CI

LacI

6

shift end shift begin

Shift (mm)

Time (hrs)

60

GFP

R

1

4 2 0 0.01

0.1 LacI decay, γL (min-1 )

1

Applications  Biomedical

Tissue regeneration Cancer therapy Other genetic diseases Artificial immune system

 Environmental

Environmental remediation Biosensing Energy production

 Biomolecular synthesis and fabrication Optimized drug synthesis Molecular scale device fabrication

 Improved understanding of natural phenomenon