Download IGEM_Chiba_2006

We fought iGEM at:
JAPAN
Farthest Place Award?
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
Toru Karasawa
Yohei Tashiro
Tomoki Morijiri
Goal ~a bacterial movie
1. Trap the E.coli by light
2. Make a light mask
-> picture
3. Move the mask
-> movie
Goal ~a bacterial movie
1. Trap the E.coli by light
2. Make a light mask
-> picture
3. Move the mask
-> movie
Goal ~a bacterial movie
1. Trap the E.coli by light
2. Make a light mask
-> picture
3. Move the mask
-> movie
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)
Light
OmpC
Tsr
tumble
Tsr
run
Microscope Assay
Test Construction
Cell: Tsr strain
pSB1A2-based
Ara ON
araP
Tsrtumble
Method
‘Tumble’ media: Ara ~0.3%
‘Run’ media: Ara 0%
observe 10sec.each.
Tsr-tumble expression
significantly slowed down
E.coli.
Ara OFF
Capillary Assay
Test Construction
Cell: Tsr strain
pSB1A2-based
Method
ＶＳ
araC
Tsr
tumble
ｔｅｔＰ
ｍＲＦＰ
Results
LB
Tsr-tumble expression seems
to slow down E.coli .
(Adler,J.Gen.MicroBiol,74;77-91,1973)
Swarm Plate Assay
Test Construction
Cell: Tsr strain
Plasmid :pSB1A2
araP
Swarm Plate contains:
Tsrtumble
: 2 % LB Broth
: 0.25 % agar
: H20
:Ampicilin
Ara 0%
Ctrl
araP- tsr-tumble
Ara 0.1%
Tsr-tumble expression seems to
slowed down E.coli… a bit …
A little smaller?
Conclusion
We learned
• Tsr-tumble really change the swimming behavior of
E.coli.
Yet to be done
• Assemble the full
construct.
Light
OmpC
• Connect with the light sensor.
Tsr
tumble
and draw a picture…
then make a bacterial movie!
Tsr
run
4 Projects
Swimmy Bacteria
…stops with light
Aromatic E.coli
…smells good
Balloon E.coli
…grows like a balloon
E.Colock
…self-cycling
Aromatic
E.coli
Switching of aroma
K.Suzuki
D.Abe
K.Nakajima
M.Ikezumi
S.Fukushima
Our aroma ; terpenes
1.ubiquitous
2.similarity and diversity
3.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 .
・Curiosity; to what
extent can bug swell
without system failure
(Can it be observable
by naked eyes?)
・FACS sorting easier
・More protein/cell
Yuki Asari
Tomoko Kaneda
Masaki Mihashi
Kayo Yasuno
Construct
ftsZ : synthesizing septum
△ftsZ
rodAmut
rodA : keeping cell rod shape
(Begg, k. j. et al., J. bacteriol., 163, 615-622 (1985).)
Light OFF
ON
OmpC
ftsZ
CI
OFF
CIP
ON
OFF
Light ON
Light sensor
(Anselm, L. et al., Nature., 438, 441-442 (2005). )
rodAmut
Experiments & Results
• Development of rodAmut
50
△minC
△minC(rodAmut)
late [%]
40
30
20
10
5 μm
5 μm
0
0
△minC
△minC(rodAmut)
10
20 30 40
length [μm]
50
Experiments & Results
•Construction of CI inverter
OmpC
CI
OmpR(+)
CIP
RFP
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 Akinori
Matuno Shinya
Okuyama Shinpei
E.Colock
Installing Kai system,
E.Coli starts oscillating.
Kai system
Kai system
Secondary output?
P
KaiA
KaiC
P
?
KaiC
P
?
KaiB
?
Protein interactions
Target site
?
E.Colock project
SasA and RpaA is
highly homologous to
EnvZ and OmoR,
respectivery.
We designed
RpaA-OmpR chimera.
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!!
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 to
Sponsors/ Supporters
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Knowledge Service
YAKUKEN-SYA
Greiner Japan
Karl Zeiss Japan
Stratagene Japan
CHIBA University
Scientific Support and Materials
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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