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2
Our Project
Cell- cell communication
PHB production
3
←Juliet
Romeo→
Scene1 Fall in love
Scene2 Juliet’s deathlike sleep
Scene3 Romeo’s Suicide
Scene4 Juliet’s Suicide
Romeo meets and falls in love with Juliet.
Romeo commits suicide in response to
Juliet’s deathlike sleep.
Juliet commits suicide after Romeo’s suicide. 4
Juliet pretends to be dead.
Scene1 Fall in love
5
Scene2 Juliet’s deathlike sleep
Scene3 Romeo’s Suicide
Scene4 Juliet’s Suicide
Juliet commits suicide after Romeo’s suicide.
Juliet pretends to be dead.
Romeo commits suicide in response to
Juliet’s deathlike sleep.
Romeo meets and falls in love with Juliet.
6
Scene1 Fall in love
Scene2 Juliet’s deathlike sleep
Scene3 Romeo’s Suicide
Scene4 Juliet’s Suicide
Juliet commits suicide after Romeo’s suicide.
Juliet pretends to be dead.
Romeo commits suicide in response to
Juliet’s deathlike sleep.
Romeo meets and falls in love with Juliet.
7
Scene1 Fall in love
Scene2 Juliet’s deathlike sleep
Scene3 Romeo’s Suicide
Scene4 Juliet’s Suicide
Juliet commits suicide after Romeo’s suicide.
Juliet pretends to be dead.
Romeo commits suicide in response to
Juliet’s deathlike sleep.
Romeo meets and falls in love with Juliet.
8
A.Subsystem & circuit design B.Characterization of new parts & devices C.Modeling
Cell-cell communication project
9
A.Subsystem & circuit design B.Characterization of new parts & devices C.Modeling
Cell-cell communication project
Scene1 Fall in love
Ⅰ-Positive feedback system
Scene2 Juliet’s deathlike sleep
Ⅱ-Band detect system
Scene3 Romeo’s Suicide
Ⅲ-Communication dependent inverter
Subsystems & Circuit design
Scene4 Juliet’s Suicide
Ⅲ-Communication dependent inverter
LuxI
Lysis LacI
LasR
Romeo
LacI
TetR LuxR
Lysis
LasI
Juliet
How do they play Romeo and Juliet?
LuxI
Lysis LacI
LacI
TetR LuxR
Lysis
LasI
LuxI
LasI
LasR
Scene1 Romeo meets and falls in love with Juliet.
Ⅰ-Positive feedback system
Juliet
Romeo
LacI
LacI
TetR LuxR
LuxI
LasI
LasR
Lysis
Lysis
Scene1 Romeo meets and falls in love with Juliet.
Ⅰ-Positive feedback system
Juliet
Romeo
LacI
LacI
TetR LuxR
LasR
LuxI LuxI
LasI LasI
Lysis
Lysis
Scene1 Romeo meets and falls in love with Juliet.
Ⅰ-Positive feedback system
Juliet
Romeo
LacI
LacI
TetR LuxR
LasR
LuxI
LasI LasI
LuxI
Lysis
Lysis
Scene2 Juliet pretends to be dead.
Ⅱ-Band detect system
Juliet
Romeo
Input=C6
LacI
LacI
LuxR
LasR
LuxI
LasI
LuxI
LasI
Lysis
Lysis
TetR
Scene2 Juliet pretends to be dead.
Ⅱ-Band detect system
Juliet
Romeo
Input=C6
LacI
LacI
LuxR
LasR
LuxI
LasI
LuxI
LasI
Lysis
Lysis
TetR
Scene2 Juliet pretends to be dead.
Ⅱ-Band detect system
Juliet
Romeo
Input=C6
LacI
LacI
LuxR
LasR
LuxI
LasI
LuxI
Lysis
Lysis
TetR
Scene3 Romeo commits suicide in response to Juliet’s deathlike sleep.
Ⅲ-Communication dependent inverter
Juliet
Romeo
LacI
LacI
LuxR
LuxI
LasI
Lysis
Lysis
TetR
Lysis
LasR
Scene3 Romeo commits suicide in response to Juliet’s deathlike sleep.
Ⅲ-Communication dependent inverter
Juliet
Romeo
LacI
LacI
LuxR
LasR
LuxI
LasI
Lysis
TetR
Lysis
Scene3 Romeo commits suicide in response to Juliet’s deathlike sleep.
Ⅲ-Communication dependent inverter
Juliet
Romeo
LacI
LacI
LuxR
LasR
LuxI
LasI
Lysis
TetR
Lysis
Scene4 Juliet commits suicide after the death of Romeo.
Ⅲ-Communication dependent inverter
Juliet
Romeo
LacI
LacI
LasR
LuxI
LasI
Lysis
TetR
Lysis
LuxR
Scene4 Juliet commits suicide after the death of Romeo.
Ⅲ-Communication dependent inverter
Juliet
Romeo
LacI
LacI
LasR
LuxI
LasI
Lysis
TetR
Lysis
LuxR
Lysis
Scene4 Juliet commits suicide after the death of Romeo.
Ⅲ-Communication dependent inverter
Juliet
Romeo
LacI
LacI
LuxR
LasR
LuxI
LasI
Lysis
TetR
Lysis
Scene4 Juliet commits suicide after the death of Romeo.
Ⅲ-Communication dependent inverter
Juliet
Romeo
25
A.Subsystem & circuit design B.Characterization of new parts & devices C.Modeling
Cell-cell communication project
Characterization of new parts and
devices for cell-cell communication
26
Ⅰ-positive feedback system
– C6 dependent C12 production
– C12 dependent C6 production
– positive feedback assay
Ⅱ-Band detect system
– Lux/Tet hybrid promoter assay
Ⅲ-Communication dependent inverter
– C6 dependent inverter
– C12 dependent inverter
Scene1 Romeo meets and falls in love with Juliet.
Ⅰ-Positive feedback system
Ⅰ-1.C6 dependent
C12 production module
Ⅰ-2.C12 dependent
C6 production module
Ⅰ-3.positive feedback assay
LuxI
lysis LacI
LasR
LacI
TetR LuxR
Lysis
LasI
C6-dependent C12 production
28
Juliet
Romeo
Plas-GFP
Ptrc Plas
lasR gfp
Positive control Negative control
現状でーた ９月１１日
C6 C12
Ptet Plux luxR lasI
Sender Reporter
C6 －＋ －＋ －＋ －＋ －＋ －＋
None Promoter for LasI
In Sender E.coli Plux Plux None None Plux
Las reporter
0
400
800
1200
Flu
ore
sce
nce
Inte
nsity (
a.u
.)
New Part
Plux-LasI K934022
20 folds
C6-dependent C12 production
pSB6A1 pSB3K3 pSB6A1 pSB3K3
Scene1 Romeo meets and falls in love with Juliet.
Ⅰ-Positive feedback system
Ⅰ-1.C6 dependent
C12 production module
Ⅰ-2.C12 dependent
C6 production module
Ⅰ-3.positive feedback assay
LuxI
lysis LacI
LasR
LacI
TetR LuxR
Lysis
LasI
31
Juliet
Romeo
C12-dependent C6 production
0
500
1000
1500
2000
3OC12HSL
Ptet Plux luxR gfp
Ptrc Plas lasR luxI
Sender Reporter 3OC6HSL
Plux-GFP Positive control Negative control
9/19生データ
Improved Part
Plas-LuxI K934012
Lux reporter
C12 －＋ －＋ －＋ －＋ －＋ －＋
None Promoter for LuxI
In Sender E.coli Plas Plas None None Plas
Flu
ore
sce
nce
Inte
nsity (
a.u
.)
110 folds
C12-dependent C6 production
pSB6A1 pSB3K3 pSB6A1 pSB3K3
Scene1 Romeo meets and falls in love with Juliet.
Ⅰ-Positive feedback system
Ⅰ-1.C6 dependent
C12 production module
Ⅰ-2.C12 dependent
C6 production module
Ⅰ-3.positive feedback assay
Scene1 Romeo meets and falls in love with Juliet.
Ⅰ-Positive feedback system
Ⅰ-1.C6 dependent
C12 production module
Ⅰ-2.C12 dependent
C6 production module
Ⅰ-3.positive feedback assay
LuxI
lysis LacI
LasR
LacI
TetR LuxR
lysis
LasI
positive feedback assay
35
Juliet
Romeo
lasR luxI
Ptet Plux luxR lasI
Ptrc Plas
positive feedback assay
Plux-LasI cell
Plas-LuxI cell
pSB6A1 pSB3K3
pSB6A1 pSB3K3
Positive feedback system is working!!
0
500
1000
1500
Las reporter
Lux reporter
positive control
negetive controlFlu
ore
sce
nce
Inte
nsity (
a.u
.)
None None C6 C12 C6 C12 Induction Plux-LasI cell &
Plas-LuxI cell
ΔP-LasI cell &
ΔP-LuxI cell
lasR luxI
Ptet Plux luxR lasI
Ptrc Plas
pSB6A1 pSB3K3
pSB6A1 pSB3K3
Scene2 Juliet pretends to be dead.
Ⅱ-Band detect system Input=C6
Ⅱ. Lux/Tet hybrid promoter assay
LuxI
lysis LacI
LasR
LacI
TetR LuxR
Lysis
LasI
Lux/Tet hybrid promoter
39
Juliet
Romeo
40
Band detect system Previous system (Basu et al. 2005) Our system
regulatory proteins 4 2
New Biobrick Part
K934024 & K934025
Tokyo_Tech 2012
-35 -10
LuxR- operator
TetR- operator
-35 -10
LuxR- operator
TetR- operator
TetR-
operator
案1 パーツ比較
Previous Biobrick Part
K176000
USTC 2009
We improved Lux/Tet hybrid promoter
No data
30 folds 140 folds
42
A.Subsystem & circuit design B.Characterization of new parts & devices C.Modeling
Cell-cell communication project
Can E.coli perform
“Romeo and Juliet"?
43
44
A.Subsystem & circuit design B.Characterization of new parts & devices C.Modeling
Cell-cell communication project
45
Whether our circuit can reproduce “Romeo and Juliet”
46
Whether our circuit can reproduce “Romeo and Juliet”
47
Whether our circuit can reproduce “Romeo and Juliet”
48
Whether our circuit can reproduce “Romeo and Juliet”
49
Whether our circuit can reproduce “Romeo and Juliet”
50
Whether our circuit can reproduce “Romeo and Juliet”
51
0
2
4
6
8
10
12
14
0 10 20 30
con
cen
trat
ion
[nM
]
time[h]
Romeo signal
Juliet signal
Whether our circuit can reproduce “Romeo and Juliet”
53
Modeling shows
feasibility of the scenario
Scene1 Fall in love
Romeo meets and falls in love with Juliet.
0
5
10
15
0 10 20 30
time[h]
－Romeo signal －Juliet signal
Con
ce
ntr
ation[n
M]
54
Juliet pretends to be dead.
Scene2 Play dead Modeling shows
feasibility of the scenario
0
5
10
15
0 10 20 30
time[h]
－Romeo signal －Juliet signal
Con
ce
ntr
ation[n
M]
55
Romeo commits suicide.
Scene3 Romeo Suicide Modeling shows
feasibility of the scenario
0
5
10
15
0 10 20 30
time[h]
－Romeo signal －Juliet signal
Con
ce
ntr
ation[n
M]
0
5
10
15
0 10 20 3056
Juliet commits suicide.
Scene4 Juliet Suicide Modeling shows
feasibility of the scenario
time[h]
－Romeo signal －Juliet signal
Con
ce
ntr
ation[n
M]
Death without love
Death after love
Simulations with various α parameters
Shy Ｊｕｌｉｅｔ can not experience true love
2700
1800 18 180000
27
270000
0
2
4
6
8
10
12
14
0 10 20 30
co
nce
ntr
ati
on
[nM
]
time[h]
Normal Juliet
Shy Juliet
αR
(Max expression rate of LuxI)
αJ
(Max e
xpre
ssio
n r
ate
of
LasI)
0
5
10
15
0 10 20 3058
A. in CO-culture system
B. in separately cultured
cell system
0
5
10
0 10 20 30
time[h]
co
ncen
tration[n
M] －Romeo signal
－Juliet signal
Positive feedback actually occurs
in the whole system
－Romeo signal
－Juliet signal
time[h]
0
5
10
15
0 10 20 3059
Band detect system works
in the whole system
0
10
20
30
0 5
0
10
20
0 5 10
Input (Romeo signal) [nM]
Outp
ut
(Ju
liet
LasI)
[nM
]
Input (Juliet signal) [nM]
Outp
ut
(Rom
eo L
uxI)
[nM
]
Con
ce
ntr
ation[n
M]
time[h]
－Romeo signal －Juliet signal
Achievement
Ⅰ. positive feedback system
Dry: guidance for wet experiments
Wet: signal increase by cell-cell communication
Ⅱ. Band detect system
Wet: Hybrid promoter
Dry: demonstration of feasibility
60
Ⅲ. Modeling
feasibility confirmation of
whole story 0
5
10
15
0 10 20 30time[h]
Romeo signalJuliet signal
60
0
10
20
0 5 10
Input (Romeo signal) [nM]
Ou
tpu
t
(Ju
liet L
asI)
[n
M]
Concentr
atio
n[n
M]
There is a famous
scene of "Romeo and Juliet”.
61
62
Bioplastic, PHB
(Poly-3-hydroxybutyrate)
2 Acetyl-CoA
Acetoacetyl-CoA
(R)-3-hydroxybutyl-CoA
Poly[(R)-3-hydroxybutyrate]
PhaA
PhaB
PhaC
NADPH
NADP+
Glucose
PhaA PhaB PhaC
Enzymes from Ralstonia eutropha
CoA
CoASH
n
Pwt RBS
phaC phaA phaB
63
BBa_K934001 Difference between cells
storing PHB
and cells
not storing PHB
PHB production
PHB
New Biobrick Part
K934001
Parts Submitted to the Registry
pha C1-A-B1 BBa_K934001
Plas-LuxI BBa_K934012
Plux-LasI BBa_K934022
Plux/tet-GFP BBa_K934025
PlasI-LacI BBa_K934016
Plux-LacI BBa_K934026
64
65
Attribution
・The ideas were proposed
by T.S( "Romeo and Juliet by E.coli Cell-cell communication“ )
and T.N("PHB production").
・The parts submitted to the registry were constructed by T.H
and N.T.
・Assays in "Romeo and Juliet by E.coli Cell-cell communication“
were carried out by M.M, X.T and S.L.
・Experiments in “PHAs production” were carried out
by L.J and T.N.
Extension of our projects
Cell-cell communication
Experimental model
for ecosystem
PHB production
Water- repellent
66
(Balagadde et al. 2008)
Membrane
Bioplastic
67
Human practice
1.Plan an Imaginary synbio project
2.Get Peer-Review
the project from public points of view
3.Improve the project
The General Public People
Pla, Do, Check ,Action PDCA cycle Propose Constructive advices
Sponsor
Aizawa Foundation
Instructors Shotaro Ayukawa
Masayuki Yamamura
Daisuke Kiga
Takeharu Tsuge
Advisors Hiroe Kawabata
Azusa Saika
Manami Hyakutake
68
Special Thanks
Professor
Hiroyuki Ohta
Eiry Kobatake
Toshiaki Mori
Miki Saijo
Kayoko Nohara
iGEM TokyoTech veteran
Yui Oshima 2009
Shinya Tahara 2009
Taichi Nakamura 2010
Thamamongood Thiprampai 2010
Mitsuhiko Odera 2010
Yuto Sugiuchi 2011
Tomoyuki Ohno 2011
Naiko hayashi 2011
Natsuki Kubo 2011 69
Member
Taku Nakayama Naoki Tsukuda
Jinglin Liu Takuo Sugiyama Shiyue Liu
Nobuaki Yasuo Xinran Tao Toshiki Hashimoto Mai Miura 70
71
Thank you
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