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We fought iGEM at:

We fought iGEM at:. JAPAN. Farthest Place Award?. GRAVEYARD. GRAVEYARD. 4 Projects. Swimmy Bacteria …stops with light Aromatic E.coli …smells good Balloon E.coli …grows like a balloon E.Colock …self-cycling. Swimmy Bacteria. Light-trapped E.coli. Maiko Furubayashi

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We fought iGEM at:

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  1. We fought iGEM at: JAPAN Farthest Place Award?

  2. GRAVEYARD GRAVEYARD

  3. 4 Projects Swimmy Bacteria …stops with light Aromatic E.coli …smells good Balloon E.coli …grows like a balloon E.Colock …self-cycling

  4. Swimmy Bacteria Light-trapped E.coli Maiko Furubayashi Toru Karasawa Yohei Tashiro Tomoki Morijiri

  5. Goal ~a bacterial movie • Trap the E.coli by light • Make a light mask -> picture • Move the mask -> movie

  6. Goal ~a bacterial movie • Trap the E.coli by light • Make a light mask -> picture • Move the mask -> movie

  7. Goal ~a bacterial movie • Trap the E.coli by light • Make a light mask -> picture • Move the mask -> movie

  8. Light OmpC Tsr tumble Tsr run Design / Parts Tsr: chemoreceptor protein for serine attractant. Truncated Tsr: makes E.coli tumlble. Truncated Tsr mutant: makes E.coli run. (Ames & Parkinson, J.Bacteriol., 176:6340-6348, 1994)

  9. araP Tsr- tumble Microscope Assay Test Construction Cell: Tsr strain pSB1A2-based Ara ON Ara OFF Method ‘Tumble’ media: Ara ~0.3% ‘Run’ media: Ara 0% observe 10sec.each. Tsr-tumble expression significantly slowed down E.coli.

  10. ｔｅｔＰ ｍＲＦＰ araC Tsr tumble Capillary Assay Test Construction ＶＳ Cell: Tsr strain pSB1A2-based Method Results LB Tsr-tumble expression seems to slow down E.coli . (Adler,J.Gen.MicroBiol,74;77-91,1973)

  11. araP Tsr- tumble Swarm Plate Assay Test Construction Swarm Plate contains: : 2 % LB Broth : H20 : 0.25 % agar :Ampicilin Cell: Tsr strain Plasmid :pSB1A2 Ara 0% Ctrl araP- tsr-tumble A little smaller? Ara 0.1% Tsr-tumble expression seems to slowed down E.coli… a bit …

  12. Light OmpC Tsr tumble Tsr run Conclusion We learned • Tsr-tumble really change the swimming behavior of E.coli. Yet to be done • Assemble the full construct. • Connect with the light sensor. and draw a picture… then make a bacterial movie!

  13. 4 Projects Swimmy Bacteria …stops with light Aromatic E.coli …smells good Balloon E.coli …grows like a balloon E.Colock …self-cycling

  14. Aromatic E.coli Switching of aroma K.Suzuki D.Abe K.Nakajima M.Ikezumi S.Fukushima

  15. Our aroma ; terpenes 1.ubiquitous 2.similarity and diversity 3.aditional values; -anti insecticides -pain relief / relaxation -anti-inflammatory

  16. Construct ↓

  17. Result

  18. Future

  19. 4 Projects Swimmy Bacteria …stops with light Aromatic E.coli …smells good Balloon E.coli …grows like a balloon E.Colock …self-cycling

  20. The Balloon E. coli The light grow the E. coli . ・Curiosity; to what extent can bug swell without system failure (Can it be observable by naked eyes?) ・FACS sortingeasier ・More protein/cell Yuki Asari Tomoko Kaneda Masaki Mihashi Kayo Yasuno

  21. OFF ON ON OFF Construct ftsZ : synthesizing septum △ftsZ rodAmut rodA : keeping cell rod shape (Begg, k. j. et al., J. bacteriol., 163, 615-622 (1985).) Light OFF OmpC ftsZ CI CIP rodAmut Light ON Light sensor (Anselm, L. et al., Nature., 438, 441-442 (2005). )

  22. △minC △minC(rodAmut) Experiments & Results • Development ofrodAmut 5 μm 5 μm

  23. OmpC CI CIP RFP Experiments & Results • Construction of CI inverter OmpR(+) OmpR(-)

  24. Discussion & Future • △minC strain formed noodle-like shape. • We got the rodAmut that make E. coli spherical. • RodA expression in △minC strain resulted in small and spherical shape. • Inverter constructed. OmpR dependency was conformed.

  25. 4 Projects Swimmy Bacteria …stops with light Aromatic E.coli …smells good Balloon E.coli …grows like a balloon E.Colock …self-cycling

  26. E.colock Self-cycling-E.coli Sugaya Sari Katabami Akinori Matuno Shinya Okuyama Shinpei

  27. E.Colock Installing Kai system, E.Coli starts oscillating.

  28. P KaiA KaiC KaiC P KaiB P Kai system Kai system Secondary output? ? ? Target site ? ? Protein interactions

  29. E.Colock project SasA and RpaA is highly homologous to EnvZ and OmoR, respectivery. We designed RpaA-OmpR chimera.

  30. … … Chimera Design rpaA tandem chimera ompR excite PCR We used 6 excite primers on each RpaA and OmpR， It can make 6 ×6 = 36 different chimeras!!

  31. Next Step HK RR

  32. As an iGEM Rookie We learned • Summer is short • Abstraction: Even working at the system level, we still need minimum knowledge/experience on the parts.

  33. Thanks to Sponsors/ Supporters • Knowledge Service • YAKUKEN-SYA • Greiner Japan • Karl Zeiss Japan • Stratagene Japan • CHIBA University Scientific Support and Materials • Prof. J.S. Perkinson (U Utah) • Prof. Takayuki Kohchi (Kyoto Univ) • Prof. Gisho Honda (Kyoto Univ) • Prof. Jay D. Keasling (Berkeley) • Dr. Yasuo Yoshikuni(Berkeley) • Prof. Rodney Croteau (WSU) • Prof. Pichersky (U Michigan) • Prof. Hideo Iwasaki (Waseda Univ) • Prof. Norhiko Misawa (Marine Biotech. Institute) • Dr. Anselm Levskaya (UCSF) • Prof. Chris Voigt (UCSF) • Prof. Takashi Karatsu (Chiba Univ) • Prof. Kyoichi Saito (Chiba Univ) • KEIO Collections • Babysitting • Reshma Shetty • Daisuke Umeno

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