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