TuBe or not TuBe? 1 dimanche 6 novembre 2011 2 dimanche 6 - - PowerPoint PPT Presentation

TuBe or not TuBe?

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Discovery of a new communication channel between cells

Early 2011... Exciting news came out:

BACTERIAL NANOTUBES

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Discovery of a new communication channel between cells

Early 2011... Exciting news came out:

Can we characterize them ? BACTERIAL NANOTUBES

What could a synthetic biology approach bring to this problem?

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TuBe or not TuBe?

harnessing bacterial nanotubes by and for synthetic biology

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Harnessing the possibilities of the nanotube network

Factory Amorphous computing Pattern formation

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Back to reality

Dubey, G.P . & Ben-Yehuda, S. Intercellular Nanotubes Mediate Bacterial Communication. Cell (2011).

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Back to reality

Dubey, G.P . & Ben-Yehuda, S. Intercellular Nanotubes Mediate Bacterial Communication. Cell (2011).

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Back to reality

Red arrows: gfp- gaining fluorescence

CmR + KanR

Dubey, G.P . & Ben-Yehuda, S. Intercellular Nanotubes Mediate Bacterial Communication. Cell (2011).

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Appearance of GFP in originally gfp– cells

Confirmation of GFP transfer (from B. subtilis 3610 gfp+ to 3610 gfp– strains) t = 0 min t = 45 min

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CmR KanR

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CmR +KanR

• B. subtilis strains on

LBA + Cm & Kan plate

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Filter

KanR expressing GFP CmR Fluorescence image

Verification of published results Additionnal control experiment: Separating cells by filter

Alternative explanation for antibiotic resistance transfer

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Episode 2

BACTERIAL NANOTUBES

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Links between teams: collaboration map

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JAPAN

USA

ASIA EUROPE AMERICA

JAPAN

Paris

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Links between teams: collaboration map

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JAPAN What do iGEMers share?

USA

ASIA EUROPE AMERICA

JAPAN

Paris

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Assisted diffusion model through nanotubes

Cell 2 Cell 1

Modeling results: Time < 1µs Volume transfer ≈ 0.1%

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Assisted diffusion model through nanotubes

Cell 2 Cell 1

Modeling results: Time < 1µs Volume transfer ≈ 0.1%

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Passive diffusion model through nanotubes

Molecule name T7 tRNA insulin GFP glucose 1st molecule transfer (s) 4.46E-2 8.59E-1 7.43E-3 5.06E-2 2.89E-4

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Amplified & robust detection of nanotube Amplified & robust detection of nanotube communication

Emitter

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Amplified & robust detection of nanotube Amplified & robust detection of nanotube communication

Emitter Amplifier Receptor

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Amplified & robust detection of nanotube Amplified & robust detection of nanotube communication

Emitter Amplifier Receptor Reporter

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Amplified & robust detection of nanotube Amplified & robust detection of nanotube communication

Emitter Amplifier Receptor Reporter

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Amplified & robust detection of nanotube Amplified & robust detection of nanotube communication

Emitter Amplifier Receptor Reporter

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Amplified & robust detection of nanotube Amplified & robust detection of nanotube communication

Emitter Amplifier Receptor Reporter Emitter transient signal

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Amplified & robust detection of nanotube Amplified & robust detection of nanotube communication

Emitter Amplifier Receptor Reporter Receiver response Emitter transient signal

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Our designs

YFP concentrator Sporulation induction by KinA λ switch ComS diffusion system

Positive feedback loop Bistable switches Concentrator

T7 RNA polymerase diffusion tRNA Amber

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T7 pol. amber

tRNA amber diffusion Emitter

tRNA RFP T7 GFP

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T7 pol. amber

mRNA

tRNA amber diffusion Emitter

tRNA RFP T7 GFP

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T7 pol. amber

mRNA

tRNA amber diffusion Emitter Receptor

tRNA RFP T7 GFP

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T7 pol. amber

mRNA

tRNA amber diffusion Emitter Receptor Amplifier

tRNA RFP T7 GFP

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tRNA amber characterization in E.coli

GFP amber + tRNA amber GFP amber

IPTG

GFP Amber tRNA Amber

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T7 polymerase amber characterization in E.coli

2,75 5,5 8,25 11

Fluorescence/OD GFP T7 amber pT7 pVeg GFP pT7 GFP T7 amber pT7 pVeg pHs tRNA Amber

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YFP concentrator Emitter Receptor + Concentrator

TetR-YFP TetO array

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YFP concentrator Emitter Receptor + Concentrator

TetR-YFP TetO array

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YFP concentrator: characterization in E.coli

TetR-YFP/TetO Arrows:Bright YFP foci TetR-YFP/TetO Red:ibpA-mCherry Green: tetR-YFP spot TetR-YFP

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Testing nanotubes formation: YFP concentrator

Mix B.Subtilis TetR-YFP/B.Subtilis TetO Mix E.coli TetR-YFP/B.Subtilis TetO

Emitter + Receiver No TetO foci observed after dozens of repeats

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T7 autoloop design

T7 pol

Autoloop

Expression of GFP as monitor pT7 T7 pol GFP

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T7 autoloop design

T7 pol

Autoloop

Expression of GFP as monitor pT7 T7 pol GFP

External input

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T7 autoloop design

T7 pol

Autoloop

Expression of GFP as monitor pT7 T7 pol GFP

Autoloop response External input

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T7 autoloop characterization in E.coli

T7 autoloop in T7+ E.coli cells T7 autoloop in T7– B.subtilis Memory of autoloop-amplified signal

• ver time in E.coli

Fluorescence/OD Time(min)

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T7 RNA polymerase diffusion design

• B. subtilis

pT7 T7 pol GFP T7 pol

Autoloop

Expression of GFP as monitor

• B. subtilis

IPTG Induction

pHS T7 pol RFP

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T7 diffusion design modeling

Genetic network model

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Delayed differential equations

GFP T7 autoloop mRNA T7 pol from emitter

Time Number of molecules

vfvr

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Emitter/Receiver mix T7 RNA polymerase diffusion experiments

TRANS RFP GFP

Plasmidic

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Emitter/Receiver mix T7 RNA polymerase diffusion experiments

TRANS RFP GFP

Plasmidic Chromosomal

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Microfluidic chambers for nanotube formation

Mondragón-Palomino, O., Danino, T., Selimkhanov, J., Tsimring, L. & Hasty, J. Entrainment of a population of synthetic genetic oscillators. Science (2011).

Why use microfluidic device?

Monolayer Exponential phase Long-time observation

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Mix of B.subtilis

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Achievements

✓Successfully reproduced and improved the original experiments, proposed

an alternative hypothesis.

✓Designed, modeled and characterized 6 emitter/receivers in E.coli and B.subtilis. ✓Developed 2 original computational diffusion models accounting for

transport through nanotubes.

✓Provided proofs of principle of 5 working emitter/receiver devices. ✓Created 49 new BioBricks and characterized 25 BioBricks for B. subtilis. ✓ Collaboration:

• Grenoble iGEM team for the human practice
• Fatih Turkey team for the rewriting of the B.Subtilis page of the Parts Registry
• Dundee, Edinbourgh, Freibourg, Pekin

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Conclusion

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Conclusion

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Smaller molecules Microfluidic conditions Statistical methods EM microscopy

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TuBe or not TuBe?

The question remains!

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The mentors Ariel Lindner, Yifan Yang, Aleksandra Nivina, Antoine Decrulle,Raphaël Pantier, Thomas Lombès Hosting laboratory

The Team

Hovannes Agopyan Adrien Basso-Blandin Ouriel Caën Baptiste Couly Laura Da Silva Mathias Toulouze Kévin Yauy Camille Huet de Froberville Edward Kwarteng Danyel Lee Adrien Lhomme-Duchadeuil Oleg Mikhajlov Babak Nichabouri Cyrille Pauthenier Axel Séguret

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A special thanks to the Grenoble’s iGEM team for our great collaboration!

Acknowledgments

• M. Elowitz, Caltech
• L. A. Sonenshein, Tufts University

J. V. Veening, Gröningen Usiversity

• H. Putzer and C. Condon, from IBPC

P .Dubey and S.Ben-Yehuda, Hebrew University of Jerusalem

• S. Serror, Orsay University
• D. Lane, Toulouse II University

P . Bassereau, Institut Curie

• Y. Chai, Harvard University

Help from labs all around the world

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% of teams with HP projects

Total number of iGEM teams Number of iGEM teams having a human practice project

Penetrance of human practice questioning within iGEM

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FACS experiments

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T7 RNA polymerase diffusion design

• B. Subtilis

pT7 T7 pol GFP T7 pol

Autoloop

Expression of GFP as monitor

• B. Subtilis

IPTG Induction

pHS T7 pol RFP

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tRNA amber design modeling

Time (s) Functional polymerases produced

Random walker model Genetic network model

T7 RNAp translated amber mRNA tRNA amber

Time (hours) Number of molecules

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Check up: where are we now?

Our project: 15 working and characterized parts submitted 25 50 Before 2011 With our contribution

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Working & Available biobricks for B. subtilis

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Sporulation induction by KinA

pEps SpoA GFP KinA KinA Phosphorelay pHS

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Hypothesis for nanotube formation

Local instability Bulge formation Nanotube formation

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Testing nanotubes formation: YFP concentrator

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Control TetR-YFP B.Subtilis Mix B.Subtilis TetR-YFP/B.Subtilis TetO Control TetR-YFP E.coli Mix E.coli TetR-YFP/B.Subtilis TetO

Emitter only Emitter + Receiver

E.coli/B.subtilis B.subtilis/B.subtilis

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