TuBe or not TuBe? dimanche 6 novembre 2011

Transcription

1 TuBe or not TuBe? 1

2 2

3 Early Exciting news came out: Discovery of a new communication channel between cells BACTERIAL NANOTUBES 2

4 Early 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

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6 TuBe or not TuBe? harnessing bacterial nanotubes by and for synthetic biology 3

7 Harnessing the possibilities of the nanotube network Factory Pattern formation Amorphous computing 4

8 Back to reality Dubey, G.P. & Ben-Yehuda, S. Intercellular Nanotubes Mediate Bacterial Communication. Cell (2011). 5

9 Back to reality Dubey, G.P. & Ben-Yehuda, S. Intercellular Nanotubes Mediate Bacterial Communication. Cell (2011). 5

10 rescence microscopy 15 hr after plating, when small colonies were visible. The dashed line indicates the border between the two populations. (a) Phase contrast image (blue). (b) GFP fluorescence image (green). (c) Overlay of phase and GFP fluorescence images. The scale bar represents 10 mm. (B) Average fluorescence intensity of the gfp! population (as indicated in Aa) as a function of the distance from the gfp+ population. The gfp! region was divided into identical sub-regions and the average fluorescence signal was defined in arbitrary units (AU). (C) Exponentially growing PY79 (gfp!) and SB444 (gfp+) cells were mixed, plated on an LB agarose pad, and incubated in a temperature controlled chamber at 37" C. Cells were visualized by time-lapse fluorescence microscopy and phase contrast (blue) and fluorescence (green) images collected at 10 min intervals. Select overlay images are shown from the following time points: (a) t0 min, (b) t30 min, and (c) t60 min. Each pair of colored arrows (red and yellow) indicates different locations where transfer of fluorescent molecules between neighboring cells is increasing over time. Larger fields of the same Redregion arrows: are shown in Figure S1. The scale bar represents gfp-1gaining mm. fluorescence (D) Average fluorescence intensity of the gfp! cells as a function of their distance from the gfp+ cells at t0 min (light blue bars) and at t60 min (dark blue bars) of the coincubation experiment as described in (C) (see Extended R was Experimental Procedures). NoR detectible signal Dubey, G.P. & Ben-Yehuda, S. Intercellular Nanotubes Mediate Bacterial Communication. Cell (2011). measured when cells were located beyond 1mm at t0 min. Back to reality Cm + Kan 5

11 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

12 Alternative explanation for antibiotic resistance transfer 1 Verification of published results CmR KanR CmR +KanR B. subtilis strains on LBA + Cm & Kan plate 2 Additionnal control experiment: Separating cells by filter Filter KanR expressing GFP CmR Fluorescence image 7

13 BACTERIAL NANOTUBES Episode 2 8

14 Links between teams: collaboration map AMERICA 2011 JAPAN JAPAN USA ASIA Paris EUROPE 9

15 Links between teams: collaboration map AMERICA What do igemers 2011 share? JAPAN JAPAN USA ASIA Paris EUROPE 9

16 Assisted diffusion model through nanotubes Cell 1 Cell 2 Modeling results: Time < 1µs Volume transfer 0.1% 10

17 Assisted diffusion model through nanotubes Cell 1 Cell 2 Modeling results: Time < 1µs Volume transfer 0.1% 10

18 Passive diffusion model through nanotubes Molecule name 1st molecule transfer (s) T7 trna insulin GFP glucose 4.46E E E E E-4 11

19 Amplified & robust detection of nanotube Amplified & robust detection of nanotube communication Emitter 12

20 Amplified & robust detection of nanotube Amplified & robust detection of nanotube communication Receptor Amplifier Emitter 12

21 Amplified & robust detection of nanotube Amplified & robust detection of nanotube communication Receptor Amplifier Emitter Reporter 12

22 Amplified & robust detection of nanotube Amplified & robust detection of nanotube communication Receptor Amplifier Emitter Reporter 12

23 Amplified & robust detection of nanotube Amplified & robust detection of nanotube communication Receptor Amplifier Emitter Reporter 12

24 Amplified & robust detection of nanotube Amplified & robust detection of nanotube communication Receptor Amplifier Emitter Reporter Emitter transient signal 12

25 Amplified & robust detection of nanotube Amplified & robust detection of nanotube communication Receptor Amplifier Emitter Reporter Receiver response Emitter transient signal 12

26 Our designs Concentrator Positive feedback loop YFP concentrator T7 RNA polymerase diffusion trna Amber ComS diffusion system Bistable switches λ switch Sporulation induction by KinA 13

27 trna amber diffusion T7 pol. amber trna RFP T7 GFP Emitter 14

28 trna amber diffusion mrna T7 pol. amber trna RFP T7 GFP Emitter 14

29 trna amber diffusion mrna T7 pol. amber trna RFP T7 GFP Emitter Receptor 14

30 trna amber diffusion mrna T7 pol. amber trna RFP T7 GFP Emitter Receptor Amplifier 14

31 trna amber characterization in E.coli IPTG trna Amber GFP Amber GFP amber GFP amber + trna amber 15

32 T7 polymerase amber characterization in E.coli pt7 11 GFP Fluorescence/OD 8,25 5,5 2,75 0 pt7 pveg GFP T7 amber pt7 pveg phs GFP T7 amber trna Amber 16

33 YFP concentrator TetR-YFP TetO array Emitter Receptor + Concentrator 17

34 YFP concentrator TetR-YFP TetO array Emitter Receptor + Concentrator 17

35 YFP concentrator: characterization in E.coli TetR-YFP TetR-YFP/TetO Arrows:Bright YFP foci TetR-YFP/TetO Red:ibpA-mCherry Green: tetr-yfp spot 18

36 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

37 T7 autoloop design T7 pol pt7 T7 pol GFP Expression of GFP as monitor Autoloop 20

38 T7 autoloop design T7 pol pt7 T7 pol GFP Expression of GFP as monitor Autoloop External input 20

39 T7 autoloop design T7 pol pt7 T7 pol GFP Expression of GFP as monitor Autoloop Autoloop response External input 20

40 T7 autoloop characterization in E.coli T7 autoloop in T7 + E.coli cells T7 autoloop in T7 B.subtilis Fluorescence/OD Memory of autoloop-amplified signal over time in E.coli Time(min) 21

41 T7 RNA polymerase diffusion design B. subtilis IPTG Induction T7 pol pt7 T7 pol GFP Expression of GFP as monitor phs T7 pol RFP Autoloop B. subtilis 22

42 T7 diffusion design modeling Delayed differential equations Genetic network model Number of molecules GFP T7 autoloop mrna T7 pol from emitter 1h Time vfvr 2h 23

43 T7 RNA polymerase diffusion experiments Emitter/Receiver mix Plasmidic TRANS RFP GFP 24

44 T7 RNA polymerase diffusion experiments Emitter/Receiver mix Chromosomal Plasmidic TRANS RFP GFP 24

45 Microfluidic chambers for nanotube formation Why use microfluidic device? Monolayer Exponential phase Long-time observation Mix of B.subtilis Mondragón-Palomino, O., Danino, T., Selimkhanov, J., Tsimring, L. & Hasty, J. Entrainment of a population of synthetic genetic oscillators. Science (2011). 25

46 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

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48 Conclusion 27

49 Conclusion Smaller molecules Microfluidic conditions Statistical methods EM microscopy 27

50 The question remains! TuBe or not TuBe? 27

51 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 Hosting laboratory The mentors Ariel Lindner, Yifan Yang, Aleksandra Nivina, Antoine Decrulle,Raphaël Pantier, Thomas Lombès 28

52 Acknowledgments A special thanks to the Grenoble s igem team for our great collaboration! Help from labs all around the world M. Elowitz, Caltech S. Serror, Orsay University L. A. Sonenshein, Tufts University D. Lane, Toulouse II University J. V. Veening, Gröningen Usiversity P. Bassereau, Institut Curie H. Putzer and C. Condon, from IBPC Y. Chai, Harvard University P.Dubey and S.Ben-Yehuda, Hebrew University of Jerusalem 29

53 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

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55 FACS experiments 32

56 T7 RNA polymerase diffusion design B. Subtilis IPTG Induction T7 pol pt7 T7 pol GFP Expression of GFP as monitor phs T7 pol RFP Autoloop B. Subtilis 33

57 trna amber design modeling Random walker model Functional polymerases produced Time (s) Genetic network model Number of molecules T7 RNAp translated amber mrna trna amber Time (hours) 19

58 Check up: where are we now? Working & Available biobricks for B. subtilis Our project: 15 working and characterized parts submitted Before 2011 With our contribution 35

59 Sporulation induction by KinA Phosphorelay phs KinA KinA peps SpoA GFP 36

60 Hypothesis for nanotube formation Local instability Bulge formation Nanotube formation 37

61 Testing nanotubes formation: YFP concentrator Control TetR-YFP E.coli Mix E.coli TetR-YFP/B.Subtilis TetO E.coli/B.subtilis Emitter only Control TetR-YFP B.Subtilis Emitter + Receiver Mix B.Subtilis TetR-YFP/B.Subtilis TetO B.subtilis/B.subtilis 13