we fought igem at:
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We fought iGEM at:
Farthest Place Award?
JAPAN
GRAVEYARDGRAVEYARD
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 FurubayashiToru KarasawaYohei TashiroTomoki Morijiri
Goal ~a bacterial movie1. Trap the E.coli by light2. Make a light mask -> picture
3. Move the mask -> movie
Goal ~a bacterial movie1. Trap the E.coli by light2. Make a light mask -> picture
3. Move the mask -> movie
Goal ~a bacterial movie1. Trap the E.coli by light2. Make a light mask -> picture
3. Move the mask -> movie
Design / Parts
Tsr:Tsr: chemoreceptor protein for serine atchemoreceptor protein for serine attractant.tractant.
Truncated Tsr:Truncated Tsr: makes E.coli makes E.coli tumltumlbleble..Truncated Tsr mutant:Truncated Tsr mutant: makes E.coli makes E.coli run.run.
OmpC
Light
Tsrtumble
Tsrrun
(Ames & Parkinson, J.Bacteriol., 176:6340-6348, 1994)
Microscope Assay
Tsr-tumble expression Tsr-tumble expression significantly slowed down significantly slowed down E.coli.E.coli.
araP Tsr-tumble
Cell: Tsr strainCell: Tsr strainpSB1A2-basedpSB1A2-based
Test Construction
Method‘‘Tumble’ media: Ara ~0.3%Tumble’ media: Ara ~0.3%‘‘Run’ media: Ara 0%Run’ media: Ara 0% observe 10sec.each.observe 10sec.each.
Ara ON
Ara OFF
Capillary Assay
araC Tsrtumble
Cell: Tsr strainCell: Tsr strainpSB1A2-basedpSB1A2-based
tetP mRFPVSVS
Method Results
LBLB
Tsr-tumble expression seems tTsr-tumble expression seems to slow down o slow down E.coli .E.coli .
Test Construction
(Adler,(Adler,J.Gen.MicroBiolJ.Gen.MicroBiol,74;77-91,1973),74;77-91,1973)
Swarm Plate Assay
Swarm Plate contains: : 2 % LB Broth : H20 : 0.25 % agar :Ampicilin
Ara Ara 0%0%
Ara Ara 0.1%0.1%
araP- tsr-tumblearaP- tsr-tumbleCtrlCtrl A little smaller?A little smaller?
Tsr-tumble expression seems toTsr-tumble expression seems to slowed down slowed down E.coli… a bit …E.coli… a bit …
araP Tsr-tumble
Cell: Tsr strainCell: Tsr strainPlasmid :pSB1A2Plasmid :pSB1A2
Test Construction
ConclusionWe 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!
OmpC
Light
Tsrtumble
Tsrrun
4 Projects
Swimmy Bacteria … stops with light
Aromatic E.coli … smells good
Balloon E.coli … grows like a balloon
E.Colock … self-cycling
Switching of aroma
K.SuzukiD.AbeK.NakajimaM.IkezumiS.Fukushima
Aromatic E.coli
Our aroma ; terpenes
1.ubiquitous2.similarity and diversity3.aditional values; -anti insecticides -pain relief / relaxation -anti-inflammatory
Construct
↓
Result
Future
4 Projects
Swimmy Bacteria … stops with light
Aromatic E.coli … smells good
Balloon E.coli … grows like a balloon
E.Colock … self-cycling
The Balloon E. coli
The light grow the E. coli .
Yuki AsariTomoko KanedaMasaki MihashiKayo Yasuno
(Can it be observable by naked eyes?)
・ Curiosity; to what extent can bug swell without system failure
・More protein/cell
・ FACS sorting easier
ConstructftsZ : synthesizing septum
rodA : keeping cell rod shape (Begg, k. j. et al., J. bacteriol., 163, 615-622 (1985).)
△ftsZ
rodAmut
Light OFF
Light ON
OmpC rodAmutCI CIPftsZ
ONOFF
ON OFF
Light sensor(Anselm, L. et al., Nature., 438, 441-442 (2005). )
Experiments & Results• Development of rodAmut
△minC(rodAmut)
0
10
20
30
40
50
0 10 20 30 40 50length [μ m]
late
[%]
△ minC△ minC(rodAmut)
△minC
5 μm 5 μm
•Construction of CI inverter
OmpC CI CIP RFP
Experiments & Results
OmpR(-)OmpR(+)
Discussion & Future
1. △minC strain formed noodle-like shape.
2. We got the rodAmut that make E. coli spherical.
3. RodA expression in △minC strain resulted in small and spherical shape.
4. Inverter constructed. OmpR dependency was conformed.
4 Projects
Swimmy Bacteria … stops with light
Aromatic E.coli … smells good
Balloon E.coli … grows like a balloon
E.Colock … self-cycling
E.colock
Self-cycling-E.coli
Sugaya Sari Katabami AkinoriMatuno ShinyaOkuyama Shinpei
E.Colock
Installing Kai system,E.Coli starts oscillating.
Kai system
KaiC KaiC
KaiB
KaiA
P
P
P
Secondary output?
?
?
?
?Target site
Protein interactions
Kai system
E.Colock project
We designed RpaA-OmpR chimera.
SasA and RpaA is highly homologous to EnvZ and OmoR, respectivery.
Chimera DesignrpaA
ompR
tandem chimera
excite PCR
……We used 6 excite primers on each RpaA and OmpR ,
It can make 6 ×6 = 36 different chimeras!!
Next Step
HK RR
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.
Thanks toSponsors/ 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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