Conclusion Constructed new Biobrick parts for Cell-cell communication

Accomplished Positive feedback system

Designed new Band detect system

Confirmed the whole system by modeling

Found out sensitive parameters and correlations

Constructed the first Biobrick part to synthesize Bioplastics

Attribution AcknowledgementWe are proud to say that whole project we worked on this summer

was proposed and carried out by undergraduate members of

our own team.

・The ideas were proposed

by T.S “E.coli Cell-cell communication” and T.N “P(3HB) Production”.

・The parts submitted to the registry were constructed

by T.H and N.T.

・Experiments in “Cell-cell communication” were carried out

by M.M, X.T and S.L.

・Experiments in “PHB Production” were carried out

by J.L and T.N.

・Modeling related to "Romeo and Juliet" was done

by N.Y.

In our project, we have recreated “Romeo and Juliet” vividly by Escherichia coli. To achieve the goal, we worked on two projects.

Project1: We aimed to make E.coli play “Romeo and Juliet”, and constructed Cell-cell communication system.

Project2: We succeeded in producing Bioplastics, P(3HB) , to make a rose which appears in the drama as a symbol of their love.

Abstract Project 2 : P(3HB) Production Project 2 : P(3HB) Production

JM109 with K934001 on pSB1C3, under UV.

we concentrated the cells and painted the rose

with the cells on LB agar medium including

0.5μg/ml Nile red and 20g/L glucose,

and we incubated the plate at 37℃ for 45 hours.

We successfully identified the product as P(3HB)

using Gas Chromatography/ Mass Spectrometry.

To figure out best culture condition,

we tried culturing E.coli JM109 in 10 conditions for 48h.

This is the first Biobrick part to synthesize P(3HB),

a kind of Bioplastics. In past, 8 iGEM teams challenged

to synthesize Bioplastics, but they did not succeed.

We also drew rose silhouette by the synthesis of P(3HB)

to reproduce the balcony scene of “Romeo and Juliet”.

Our new part has 3 genes coding

PhaA, B, C enzymes from

Ralstonia eutropha H16.

“Polymer concentration (g/L)” is the amount of the polymer in the medium after culturing.

This value is calculated by multiplying “Dried cells” and “Polymer content rate”.

The results showed that TB medium was much better

than LB medium to synthesize P(3HB). In the 30°C culturing

containing Glucose and Pantothenic acid Ca,

E.coli synthesized the polymer in maximum concentration.

Human Practice We participated in a science cafe as assistants. We

supported general public people who don’t specialize in

biology to discuss synthetic biology. The purpose of the

science cafe is to provide a chance both for researchers

and general citizens to reconsider the relationship

between synthetic biology and human society. During

the event, the general public people had planed many

interesting and imaginary bacteria projects about the

theme “bacteria that can realize dreams”.

Planning

Peer reviewImprovement

Reference1. Basu S, Gerchman Y, Collins CH, Arnold FH, & Weiss R (2005) A synthetic multicellular system for programmed

pattern formation. Nature 434(7037):1130-1134.

2. You L, Cox RS, Weiss R, & Arnold FH (2004) Programmed population control by cell-cell communication and

regulated killing. Nature 428:868-871.

3. Balagadde FK, et al. (2008) A synthetic Escherichia coli predator-prey ecosystem. Mol Syst Biol 4:187

4. J Biol Eng. (2008) co-fermentation strategy to consume sugar mixtures effectively Published online 2008 February

27.

5. Brenner K, You L, Arnold FH Engineering microbial consortia: a new frontier in synthetic biology Trends in

Biotechnology [2008, 26(9):483-489]

7. Akihiko K, et al. (2011) Cluster Newton Method for Sampling Multiple Solutions of an Underdetermined Inverse

Problem: Parameter Identification for Pharmacokinetics. National Institute of Informatics, NII-2011-002E.

8. Jumiarti Agus, Altered expression of polyhydroxyalkanoate synthase gene and its effect on poly[(R)-3-

hydroxybutyrate] synthesis in recombinant Escherichia coli, Polymer Degradation and Stability(2006) 91:1645-1650

9. Joanne Stubbe and Jiamin Tian, Polyhydroxyalkanoate (PHA) homeostasis: the role of the PHA synthase, 2003, Nat.

Prod. Rep.,20, 445–457.

10. Stanley D. Fowler and Phillip Greenspan, Application of Nile red, a fluorescent hydrophobic probe, for the

detection of neutral lipid deposits in tissue sections, Histochemistry & Cytochemistry(1985), vol 33.No 8, 833-836

11. Pinzon NM, Nile red detection of bacterial hydrocarbons and ketones in a high-throughput format, mBio (2011),vol

2. issue 4.e-00109-11

12. Patricia Spiekermann, A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that

accumulate polyhydroxyalkanoic acids and other lipid storage compounds, Arch Microbiol (1999), 171:73–80

13. Vladimir K. Vanag, Cross-diffusion and pattern formation in reaction–diffusion systems, Physical Chemistry

Chemical Physics(2009), vol 11.897-912

14. Pohlmann A, et al, Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16,

Nat Biotechnol(2006), 24:1257-62

Prof. Miki Saijo

Prof. Kayoko Nohara

and, Yui Oshima (2009 Tokyo_Tech team member).

Tokyo Tech Science and Engineering Communication

Science Cafe “ Bacteria make your dreams come true

(Yume wo kanaeru Saikin)”

Prof. Akihiko Konagaya

Kenta Yoshida

Prof.Hiroyuki Ohta, Prof.Eiry Kobatake,

Prof.Toshiaki Mori, Prof.Yasunori Aizawa, Prof.Yasuo Asakura

“Romeo and Juliet”by E.coli cell-cell communication

Tokyo TechTokyo TechTokyo TechTokyo TechUndergrads: Taku Nakayama, Nobuaki Yasuo, Takuo Sugiyama,

Toshiki Hashimoto, Mai Miura, Naoki Tsukuda,

Jinglin Liu, Xinran Tao, Shiyue Liu

Advisers: Hiroe Kawabata, Azusa Saika, Manami Hyakutake

Instructors: Shotaro Ayukawa, Masayuki Yamamura,

Takeharu Tsuge, Daisuke Kiga

Modeling

Project 1 : Cell-cell communication

C6

C12

Scene1 Fall in love

Scene1 is realized by Positive feedback

system between two types of E.coli.

An increase of one signal causes

the increase of the other

signal production

as their love burns.

Scene4 Juliet’s suicide

Scene4 is realized by Communication

dependent inverter in Juliet cell. In

response to Romeo’s

suicide, lysis protein

is expressed in Juliet

cell.

C6

C12

Scene3 Romeo’s suicide

Scene3 is realized by Communication

dependent inverter in Romeo cell. In

response to Juliet’s deathlike

sleep, lysis protein is

expressed in Romeo cell.

C6

C12

Scene2 Juliet’s deathlike sleep

Scene2 is represented by Band detect

system in Juliet cell. As Romeo signal

concentration reaches higher level,

Juliet cell stops producing

signals, though Juliet cell

is alive.

C6

C12

Project 1 : Cell-cell communication

Pon

luxRluxR

Plux/tet

lasIlasI

lacIlacIPlux

tetRtetRPlux

lysislysisPlac

lasRlasRPon

luxIluxIPlas

lysislysisPlac

lacIlacIPlas Romeo

Juliet

I. Signal dependent signal production

II. Time-dependent change assay

Scene1 Positive feedback system

Scene2 Band detect system

To accomplish the Positive feedback system

in the Cell-cell communication, we first

constructed and characterized two new

Biobrick parts Plux-LasI (K934022) and Plas-

LuxI (K934012).

The result confirms that the cells

containing Plux-LasI (Plux-LasI cell) produced

C12 in response to C6 induction and cells

containing Plas-LuxI (Plas-LuxI cell) produced

C6 in response to C12 induction.

Wet experiments

By co-culturing Plux-LasI cell and Plas-LuxI

cell, we confirmed the complete Positive

feedback system where the production of a

signal activates the production of the other

signal. As a trigger of the Positive feedback

system, we added the initial dose of C6 (5 nM)

to each co-culture.

As compared red solid line with blue dotted

line (both Plux-LasI cell and Plas-LuxI cell

coexist), the result indicates that the C12

production in Plux-LasI cell was activated by

initially added C6 (0-1h), whereas the C6

production in Plas-LuxI cell was not activated

till C12 production in Plux-LasI cell reached

sufficient level (1-2h). This behavior strongly

suggests the appearance of the Positive

feedback.

We designed the new Band detect

system. Compared with the Weiss

Lab’s Band detect system, our system

is constructed with fewer components.

For this, we can avoid the useless

complexity.

We characterized Lux/Tet hybrid

promoter (K934024) which is

important part for our Band detect

system. The result shows that our

Lux/Tet hybrid promoter integrated

the inputs of C6 and aTc.

We confirmed this whole system by

modeling.

regulatory proteins

4 2>

To confirm the feasibility of the whole Cell-cell

communication system, we developed an ordinary

differential equation model and simulated the

system under typical experimental conditions.

cR and cJ denote the concentration of lysis protein in Romeo

cell and Juliet cell, respectively.

NR and NJ denote the population of Romeo cell and Juliet cell.

Scene1 (yellow): Two signals increase.

Scene2 (green): As Romeo signal increases to some

extent, Juliet signal starts to decline.

Scene3 (blue): Lysis gene is expressed in Romeo cell

in response to the decline of Juliet signal, then

Romeo signal starts to decline.

Scene4 (pink): Lysis gene is expressed in Juliet cell in

response to the decline of Romeo signal, then Juliet

signal decreases further.

Parameter sensitivity and correlation analysis

We searched the feasible solution distribution of parameters that satisfies the specific time course to

reproduce either “Romeo and Juliet” or “good ending story”. Furthermore, we identified the most influential

parameters dominating the dynamics of our Cell-cell communication.

The orange/blue bandlike region of figure(2) represents the assembly of

parameter sets that satisfy the specific time course of “Romeo and Juliet”/

“Good ending story”, respectively. Then, we concluded that α2, α8 and m3

are sensitive parameters because their ranges are narrow in orange band.

We focused on Parameter α8, which may be dominant over the dynamics

of our Cell-cell communication because the overlap between two bands is

the most narrow at α8.

Furthermore, we revealed that there are relatively strong

correlation between α8 and α2 and that between α8 and m3.

Parameters α2 and m3 are related to the production and reception

of Juliet signal, respectively. We confirmed that it is important to

harmonize them with α8, which is related to the suicide system in

Juliet cell. These results have a great meaning to realize the whole

Cell-cell communication system.

In the Juliet cell, the Juliet

signal production results in

the highest level under the

particular range of Romeo

signal concentration.

Feasibility of Band detect system

(1) An example of specific time course

-10

0

10

20

30

40

50

60

0 10 20 30 40 50

sig

na

l co

nce

ntr

ati

on

[nM

]

time [h]

"Good ending story"

"Romeo and Juliet"

Feasibility of the whole system

Max expression rate of

lysis protein

Constant of proportionality

of Juliet signal synthesis

α8 :

α2 :

m3 : Threshold of Juliet signal

concentration for LacI or

LuxI production

0

4

8

12

16

20

24

0 1 2 3 4 5 6 7 8 9 10111213

Ou

tpu

t

(Ju

lie

t La

sI)[

nM

]

input(Romeo signal)[nM]

0

2

4

6

8

10

12

14

0 5 10 15 20 25 30

con

cen

tra

tio

n[n

M]

time[h]

Romeo signal

Juliet signal

0

0.1

0.2

0.3

0 0.1 0.2 0.3 0.4 0.5 0.6

Va

ria

nce

(lo

w =

se

nsi

tiv

e)

Absolute value of

correlation coefficient toward α8

m3 α2

no

rma

lize

d v

alu

e

of

pa

ram

ete

rs

α1 α9α6 α7 α10 m4 m5 m7α2 α3 α4 m8

parameter

α8 m3

(2) Feasible solution distribution Orange: “Romeo and Juliet”

Blue: “Good ending story”

(3) Parameter sensitivity and correlation

with α8 in “Romeo and Juliet”