TuBe or not TuBe? 1 dimanche 6 novembre 2011
2 dimanche 6 novembre 2011
Early 2011... Exciting news came out: Discovery of a new communication channel between cells BACTERIAL NANOTUBES 2 dimanche 6 novembre 2011
Early 2011... Exciting news came out: Discovery of a new communication channel between cells BACTERIAL NANOTUBES Can we characterize them ? What could a synthetic biology approach bring to this problem? 2 dimanche 6 novembre 2011
3 dimanche 6 novembre 2011
TuBe or not TuBe? harnessing bacterial nanotubes by and for synthetic biology 3 dimanche 6 novembre 2011
Harnessing the possibilities of the nanotube network Pattern formation Factory Amorphous computing 4 dimanche 6 novembre 2011
Back to reality 5 Dubey, G.P . & Ben-Yehuda, S. Intercellular Nanotubes Mediate Bacterial Communication. Cell (2011). dimanche 6 novembre 2011
Back to reality 5 Dubey, G.P . & Ben-Yehuda, S. Intercellular Nanotubes Mediate Bacterial Communication. Cell (2011). dimanche 6 novembre 2011
Back to reality Red arrows: gfp- gaining fluorescence Cm R + Kan R 5 Dubey, G.P . & Ben-Yehuda, S. Intercellular Nanotubes Mediate Bacterial Communication. Cell (2011). dimanche 6 novembre 2011
Appearance of GFP in originally gfp – cells t = 0 min t = 45 min Confirmation of GFP transfer (from B. subtilis 3610 gfp + to 3610 gfp – strains) 6 dimanche 6 novembre 2011
Alternative explanation for antibiotic resistance transfer Verification of published results 1 Cm R Cm R Kan R +Kan R B. subtilis strains on LBA + Cm & Kan plate Additionnal control experiment: 2 Separating cells by filter Filter Kan R expressing GFP Cm R Fluorescence image 7 dimanche 6 novembre 2011
BACTERIAL NANOTUBES Episode 2 8 dimanche 6 novembre 2011
Links between teams: collaboration map AMERICA 2011 JAPAN JAPAN USA Paris ASIA EUROPE 9 dimanche 6 novembre 2011
Links between teams: collaboration map AMERICA What do iGEMers 2011 share? JAPAN JAPAN USA Paris ASIA EUROPE 9 dimanche 6 novembre 2011
Assisted diffusion model through nanotubes Cell 1 Cell 2 Modeling results: Time < 1µs Volume transfer ≈ 0.1% 10 dimanche 6 novembre 2011
Assisted diffusion model through nanotubes Cell 1 Cell 2 Modeling results: Time < 1µs Volume transfer ≈ 0.1% 10 dimanche 6 novembre 2011
Passive diffusion model through nanotubes Molecule T7 tRNA insulin GFP glucose name 4.46E-2 8.59E-1 7.43E-3 5.06E-2 2.89E-4 1st molecule transfer (s) 11 dimanche 6 novembre 2011
Amplified & robust detection of nanotube Amplified & robust detection of nanotube communication Emitter 12 dimanche 6 novembre 2011
Amplified & robust detection of nanotube Amplified & robust detection of nanotube communication Receptor Amplifier Emitter 12 dimanche 6 novembre 2011
Amplified & robust detection of nanotube Amplified & robust detection of nanotube communication Receptor Amplifier Reporter Emitter 12 dimanche 6 novembre 2011
Amplified & robust detection of nanotube Amplified & robust detection of nanotube communication Receptor Amplifier Reporter Emitter 12 dimanche 6 novembre 2011
Amplified & robust detection of nanotube Amplified & robust detection of nanotube communication Receptor Amplifier Reporter Emitter 12 dimanche 6 novembre 2011
Amplified & robust detection of nanotube Amplified & robust detection of nanotube communication Receptor Amplifier Reporter Emitter Emitter transient signal 12 dimanche 6 novembre 2011
Amplified & robust detection of nanotube Amplified & robust detection of nanotube communication Receptor Amplifier Reporter Emitter Receiver response Emitter transient signal 12 dimanche 6 novembre 2011
Our designs Concentrator Positive feedback loop T7 RNA polymerase YFP concentrator tRNA Amber diffusion Bistable switches Sporulation ComS diffusion λ switch induction by KinA system 13 dimanche 6 novembre 2011
tRNA amber diffusion T7 pol. amber tRNA RFP T7 GFP Emitter 14 dimanche 6 novembre 2011
tRNA amber diffusion mRNA T7 pol. amber tRNA RFP T7 GFP Emitter 14 dimanche 6 novembre 2011
tRNA amber diffusion mRNA T7 pol. amber tRNA RFP T7 GFP Emitter Receptor 14 dimanche 6 novembre 2011
tRNA amber diffusion mRNA T7 pol. amber tRNA RFP T7 GFP Emitter Receptor Amplifier 14 dimanche 6 novembre 2011
tRNA amber characterization in E.coli IPTG GFP Amber tRNA Amber GFP amber + tRNA amber GFP amber 15 dimanche 6 novembre 2011
T7 polymerase amber characterization in E.coli pT7 GFP 11 Fluorescence/OD 8,25 pVeg pT7 5,5 GFP T7 amber 2,75 pHs pVeg pT7 0 GFP T7 amber tRNA Amber 16 dimanche 6 novembre 2011
YFP concentrator TetR-YFP TetO array Emitter Receptor + Concentrator 17 dimanche 6 novembre 2011
YFP concentrator TetR-YFP TetO array Emitter Receptor + Concentrator 17 dimanche 6 novembre 2011
YFP concentrator: characterization in E.coli TetR-YFP/TetO TetR-YFP/TetO TetR-YFP Arrows:Bright YFP foci Red:ibpA-mCherry Green: tetR-YFP spot 18 dimanche 6 novembre 2011
Testing nanotubes formation: YFP concentrator Emitter + Receiver Mix E.coli TetR-YFP/B.Subtilis TetO Mix B.Subtilis TetR-YFP/B.Subtilis TetO No TetO foci observed after dozens of repeats 19 dimanche 6 novembre 2011
T7 autoloop design T7 pol Expression of GFP as monitor pT7 GFP T7 pol Autoloop 20 dimanche 6 novembre 2011
T7 autoloop design T7 pol Expression of GFP as monitor pT7 GFP T7 pol Autoloop External input 20 dimanche 6 novembre 2011
T7 autoloop design T7 pol Expression of GFP as monitor pT7 GFP T7 pol Autoloop Autoloop response External input 20 dimanche 6 novembre 2011
T7 autoloop characterization in E.coli T7 autoloop in T7 + E.coli cells T7 autoloop in T7 – B.subtilis Memory of autoloop-amplified signal Fluorescence/OD over time in E.coli Time(min) 21 dimanche 6 novembre 2011
T7 RNA polymerase diffusion design B. subtilis T7 pol IPTG Induction Expression of GFP as monitor pT7 GFP T7 pol Autoloop T7 pol pHS RFP B. subtilis 22 dimanche 6 novembre 2011
T7 diffusion design modeling Delayed differential equations Genetic network model Number of molecules GFP T7 autoloop mRNA T7 pol from emitter Time 1h 2h 23 vfvr dimanche 6 novembre 2011
T7 RNA polymerase diffusion experiments Emitter/Receiver mix Plasmidic TRANS RFP GFP 24 dimanche 6 novembre 2011
T7 RNA polymerase diffusion experiments Emitter/Receiver mix Plasmidic TRANS RFP GFP Chromosomal 24 dimanche 6 novembre 2011
Microfluidic chambers for nanotube formation Mix of B.subtilis Why use microfluidic device? Monolayer Exponential phase Long-time observation 25 Mondragón-Palomino, O., Danino, T., Selimkhanov, J., Tsimring, L. & Hasty, J. Entrainment of a population of synthetic genetic oscillators. Science (2011). dimanche 6 novembre 2011
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 26 dimanche 6 novembre 2011
27 dimanche 6 novembre 2011
Conclusion 27 dimanche 6 novembre 2011
Conclusion Microfluidic conditions Smaller molecules Statistical methods EM microscopy 27 dimanche 6 novembre 2011
The question remains! TuBe or not TuBe? 27 dimanche 6 novembre 2011
The Team Hosting laboratory Hovannes Agopyan Edward Kwarteng Adrien Basso-Blandin Danyel Lee Ouriel Caën Adrien Lhomme-Duchadeuil Baptiste Couly Oleg Mikhajlov Laura Da Silva Babak Nichabouri Mathias Toulouze Cyrille Pauthenier Kévin Yauy Axel Séguret Camille Huet de Froberville The mentors Ariel Lindner, Yifan Yang, Aleksandra Nivina, Antoine Decrulle,Raphaël Pantier, Thomas Lombès 28 dimanche 6 novembre 2011
Acknowledgments A special thanks to the Grenoble’s iGEM team for our great collaboration! Help from labs all around the world S. Serror, Orsay University M. Elowitz, Caltech D. Lane, Toulouse II University L. A. Sonenshein, Tufts University P . Bassereau, Institut Curie J. V. Veening, Gröningen Usiversity Y. Chai, Harvard University H. Putzer and C. Condon, from IBPC P .Dubey and S.Ben-Yehuda, Hebrew University of Jerusalem 29 dimanche 6 novembre 2011
Penetrance of human practice questioning within iGEM % of teams with HP projects Total number of iGEM teams Number of iGEM teams having a human practice project 30 dimanche 6 novembre 2011
31 dimanche 6 novembre 2011
FACS experiments 32 dimanche 6 novembre 2011
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