Our Project - 2012.igem.org2012.igem.org/files/presentation/Tokyo_Tech.pdfScene4 Juliet’s Suicide Romeo meets and falls in love with Juliet. Romeo commits suicide in response to

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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




Juliet commits suicide after Romeo’s suicide.





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Scene1 Fall in love









7

Scene1 Fall in love









8

A.Subsystem & circuit design B.Characterization of new parts & devices C.Modeling

Cell-cell communication project

9



Scene1 Fall in love

Ⅰ-Positive feedback system


Ⅱ-Band detect system


Ⅲ-Communication dependent inverter

Subsystems & Circuit design



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.


Juliet

Romeo

LacI

LacI

TetR LuxR

LuxI

LasI

LasR

Lysis

Lysis



Juliet

Romeo

LacI

LacI

TetR LuxR

LasR

LuxI LuxI

LasI LasI

Lysis

Lysis



Juliet

Romeo

LacI

LacI

TetR LuxR

LasR

LuxI

LasI LasI

LuxI

Lysis

Lysis

Scene2 Juliet pretends to be dead.


Juliet

Romeo

Input=C6

LacI

LacI

LuxR

LasR

LuxI

LasI

LuxI

LasI

Lysis

Lysis

TetR



Juliet

Romeo

Input=C6

LacI

LacI

LuxR

LasR

LuxI

LasI

LuxI

LasI

Lysis

Lysis

TetR



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.


Juliet

Romeo

LacI

LacI

LuxR

LuxI

LasI

Lysis

Lysis

TetR

Lysis

LasR



Juliet

Romeo

LacI

LacI

LuxR

LasR

LuxI

LasI

Lysis

TetR

Lysis



Juliet

Romeo

LacI

LacI

LuxR

LasR

LuxI

LasI

Lysis

TetR

Lysis

Scene4 Juliet commits suicide after the death of Romeo.


Juliet

Romeo

LacI

LacI

LasR

LuxI

LasI

Lysis

TetR

Lysis

LuxR



Juliet

Romeo

LacI

LacI

LasR

LuxI

LasI

Lysis

TetR

Lysis

LuxR

Lysis



Juliet

Romeo

LacI

LacI

LuxR

LasR

LuxI

LasI

Lysis

TetR

Lysis



Juliet

Romeo

25



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


– Lux/Tet hybrid promoter assay


– C6 dependent inverter

– C12 dependent inverter



Ⅰ-1.C6 dependent

C12 production module

Ⅰ-2.C12 dependent


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


pSB6A1 pSB3K3 pSB6A1 pSB3K3



Ⅰ-1.C6 dependent


Ⅰ-2.C12 dependent



LuxI

lysis LacI

LasR

LacI

TetR LuxR

Lysis

LasI

31

Juliet

Romeo


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


pSB6A1 pSB3K3 pSB6A1 pSB3K3



Ⅰ-1.C6 dependent


Ⅰ-2.C12 dependent





Ⅰ-1.C6 dependent


Ⅰ-2.C12 dependent



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


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

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Juliet

Romeo

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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

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Can E.coli perform

“Romeo and Juliet"?

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Whether our circuit can reproduce “Romeo and Juliet”

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48


49


50


51

0

2

4

6

8

10

12

14

0 10 20 30

con

cen

trat

ion

[nM

]

time[h]

Romeo signal

Juliet signal


53

Modeling shows

feasibility of the scenario

Scene1 Fall in love


0

5

10

15

0 10 20 30

time[h]

－Romeo signal －Juliet signal

Con

ce

ntr

ation[n

M]

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Scene2 Play dead Modeling shows


0

5

10

15

0 10 20 30

time[h]


Con

ce

ntr

ation[n

M]

55

Romeo commits suicide.

Scene3 Romeo Suicide Modeling shows


0

5

10

15

0 10 20 30

time[h]


Con

ce

ntr

ation[n

M]

0

5

10

15

0 10 20 3056

Juliet commits suicide.

Scene4 Juliet Suicide Modeling shows


time[h]


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

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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]


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

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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”.

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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

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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

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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

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(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

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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

Documents

Our Project - 2012.igem.org2012.igem.org/files/presentation/Tokyo_Tech.pdfScene4 Juliet’s Suicide Romeo meets and falls in love with Juliet. Romeo commits suicide in response to