Regenerative medicine and tissue formation is really important in recent years.

Regenerative Medicine Tissue Formation

We need to understand mechanisms of development to regenerate our body.

Regenerative Medicine Tissue Formation

Turing Patterns exist everywhere in the natural world.

Simple mechanism makes complex Turing Pattern.

Because of the complexity of an organisms’ development, it is difficult to investigate Turing Pattern in vivo.

In order to understand mechanisms of development…

OUR GOAL: to create synthetic system forming Turing Pattern.

Two factors have three features to form Turing Pattern.

Features:1.Activation.2.Inactivation.3.Inhibitors diffuse

faster.

Two factors have three features to form Turing Pattern.

Features:1.Activation.2.Inactivation.3.Inhibitors diffuse

faster.

Two factors have three features to form Turing Pattern.

Features:1.Activation.2.Inactivation.3.Inhibitors diffuse

faster.

Two factors have three features to form Turing Pattern.

Features:1.Activation.2.Inactivation.3.Inhibitors diffuse

faster.

The demonstration of the interaction of activator and inhibitor.

Basic Strategy: System

Lateral Inhibition by E. coli and AHL reaction

Local Activation by E. coli self-reproduction

E. coli as activator and AHL as inhibitor

E. coli emits AHL and AHL kills E. coli

Lateral Inhibition: AHL is emitted outside from E. coli.

Lateral Inhibition: AHL activates Barnase.

E.coli emits AHL and AHL kills E. coli

Fig. 1 time development in basic strategy

Convergent when positive feedback is weak.

Meaning of Positive Feedback

Meaning of Positive Feedback

Meaning of Positive Feedback

Fig. 1 time development in basic strategy

Fluctuation will be 0 without positive feedback

Improved Strategy: System

Lateral Inhibition by reactions between E. coli and AHL

Local Activation by the mutual inhibition

Comparing two strategies: System

Basic Strategy Improved Strategy

Comparing two strategies: System

Basic Strategy Improved Strategy

Comparing two strategies: System

Basic Strategy Improved Strategy

The growth rate of improved strategy is smaller in the beginning.

Basic Strategy Improved Strategy

The growth rates are the same at the end.

Basic Strategy Improved Strategy

It is growth rate of growth rate that counts.

Basic Strategy Improved Strategy

Basic Strategy: solid Improved Strategy: pattern

From the same condition, it forms pattern only in improved strategy.

Turing Pattern Simulation of improved strategy.

Fig. 3 pattern by mathematical simulation

Co-activator produces Colicin and Colicin kills activator E. coli.

Local Activation: co-activator repress activator by Emitting Colicin E3.

Colicin E3 has some advantage.

1. Molar weight is large enough:

60000 ~ 61000 Da

2. Safe: only affects microbes.

Soelaiman, S., Jakes, K., Wu, N., Li, C., & Shoham, M.

(2001). Crystal structure of colicin E3: implications for cell

entry and ribosome inactivation.Molecular cell, 8(5), 1053-

1062. Figure 2. A

Local Activation: co-activator repress activator by Emitting Colicin E3.

Local Activation: activator repress co-activator because of growth competition.

Local Activation: Mutual Inhibition

Co-activator produces Colicin and Colicin kills activator E. coli

The way to achieve our goal.

Detailed plan to achieve first step.

a. Diffusion rate of E. colib. Strength of growth inhibitionc. Activity of Colicin Lysis Protein

a. Diffusion rate

Approach for calculating the diffusion rate.

2h

11h

19h

Semi-solid agar gel

The stronger expression of CheZ, the bigger diffusion rate.

Fig. 1 Motility control by CheZ

0

0.8

1.6

Strong2 CheZ Strong1 CheZ Medium CheZ CheZ - (NC)

Dif

fusi

on

Rat

e [

mm

^2/h

]

b. Strength of growth inhibition

Barnase repressed the growth of E. coli as we expected.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 20 40 60 80 100 120 140

OD

60

0

time[hr]

Plac GFP (IPTG+)Plac GFP (-)plac Barnase GFP (IPTG+)plac Barnase GFP (-)

Fig. 2 growth curve repressed by barnase

Barnase repressed the growth of E. coli as we expected.

0

0.005

0.01

0.015

0.02

0.025

Plac (IPTG-) Plac (IPTG+) plac Barnase(IPTG-)

plac Barnase(IPTG+)

Gro

wth

Rat

e [/

min

]

Fig. 3 growth rate repressed by barnase

c. Activity of Colicin Lysis Protein

Colicin Lysis Protein work together with ColicinE3 and help it secret from E. coli.

Colicin Lysis Protein worked well in E. coli.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

0 1 2 3 4 5 6 7 8

OD

60

0

Time [hr]

Plac GFP (IPTG+)Plac GFP (-)Plac ColL (+)Plac ColL (-)

Fig. 4 growth curve repressed by Col L

The way to achieve our goal.

a. Diffusion rate of E. coli

b. Strength of growth inhibition

c. Activity of Colicin Lysis Protein

Discussion

Policy and Practice: Public Engagement

May Fes.Techno Edge

Policy and Practice: Survey

New Parts : CheZ generator (BBa_K1631018-20)

Parts Characterization: Plac Barnarse –GFP(Bba_K1631012)

Parts Improvement (BBa_I7162111→BBa_K1631000)

Policy and Practice

- Public Engagement

- Intellectual Property and Synbio

References

[1]Turing, A. M. (1952). The chemical basis of morphogenesis. Philosophical Transactions of the Royal Society of London B: Biological Sciences, 237(641), 37-72.

[2] Blanchard, A. E., Celik, V., & Lu, T. (2014). Extinction, coexistence, and localized patterns of a bacterial population with contact-dependent inhibition. BMC systems biology, 8(1), 23.

AdvisorDr. James EllingerDr. Claig Hamilton

Laboratory, AdviseYouichi Wakamoto, a professor of SynBio, from University of Tokyo

OverallMother of Mr. Nakamura for her warm heartLecture room, University of Tokyo

Policy and PracticeInterviewHiroyuki Takahashi, from Leave a Nest corp. Youichi Muratomi, a professional of Intellectual Property,

from Yokohama National University Many Students from all over the world

OverallGerardo AugusutoUrbina Sánchezguests of May Festival

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THANKS FOR YOUR COOPERATION

Team UT-Tokyo 2015