1. Ninja State Switching

Team members

Adviser

Instructor

Undergraduate

Attribution

Acknowledgement

Human Practice

2. M13 Shuriken

Story

3. Farming Main Achievements

Ninja is a Japan’s ancient spy-warrior. Usually ninja disguises himself as a civilian in the public places. Once he detects samurai (assassination target), he immediately gets ready for battle. He defeats samurai with shuriken, throwing knives. In the second-life story, E. ninja starts farming in a peaceful village…

When E. civilian emits C6HSL and E. Ninja receives these molecules, the C6HSL-LuxR complex activates Plux. Then LacI starts to be expressed and represses Plac.

When E. samurai emits C12HSL and E. ninja receives these molecules, the C12HSL-LasR complex activates Plas. Then CI starts to be expressed and represses Pλ.

Shinya S. Ken S. Emma H. Yuta W. Naoki W. Tatsuhiro I. Ryota F. Masanori K. Shunta S. Sairi T. Tatsuya K. Hiroshi F. Sara O. Hengyu S. Yochiku S. Check out our website !!

Scan the QR code with your cellphone for more information.

Prof. Arisaka, Prof. Fukui, Prof. Hata, Prof. Hirota, Prof. Iwasaki, Prof. Nakamura, Prof. Ogura, Prof. Taguchi, Prof. Takinoue, Prof. Tanji, Prof. Urabe, Prof. Wachi, Prof. Whittier, Ms. Miura, Mr. Nakayama, Mr. Sugano, Mr. Tsukuda and Mr. Yasuo.

We 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 E.H. “Crosstalk circumvention”, T.I. “M13 phage” and R.F.“Plant hormone”. ・All constructions were carried out by K.S, H.F, T.K, S.S, S.T, and S.S. ・All fluorescence assays were carried out by Y.W, E.H, S.O, R.F and H.S. ・Bio assay in “Plant hormone” was carried out by R.F. ・UPLC assay in “Plant hormone” was carried out by S.S. ・All modelings were done by Y.S. and M.K. ・E. ninja and the other characters are designed by S.T.

Circumvention of crosstalk between two intercellular molecules (BBa_K1139110 new part) Part collection of M13 phage (BBa_K1139019, BBa_K1139020, BBa_K1139021) Phosphate dependent expression regulation (BBa_K1139201 improved part)

Kazuaki Amikura

Daisuke Kiga, Hiroyuki Ohta, Masayuki Yamamura, Akihiko Konagaya, Shotaro Ayukawa, Ryoji Sekine

E. ninja throws "shuriken" to attack E. samurai in response to the signal from E. samurai.

We constructed a model system for inducible phage release by induction of g2p expression [2]. We would be able to transmit desired DNA to the desired place by using our parts.

Fig. 2-a. the design of our part related to M13 phage.

Reference: [2] A Higashitani (1994)

signal

Inducible M13 phage

(The work is done by Naoki, Masanori and Tatsuhiro)

To create “Famer E. coli” In the second-life, E. ninja starts farming. Our improved part for phosphate sensing and a new part for cytokinin synthesis will create “Farmer E. coli” that synthesizes plant hormones depending on the soil environment (Fig. 3-a).

Bioassay We learned methods for quantitative analysis for cytokinin through a bioassay (Fig. 3-b).

The increase in phosphate concentration repressed the phoA promoter (Fig. 3-d). Compared to OUC-China’s phosphate sensor, our phosphate sensor shows clearer result.

Plants are reported to be in phosphate starvation (under 1 mM). Therefore, our improved part is useful. We determined parameters of phoA promoter for the induction mechanism.

We combined the phoA promoter from E. coli [3] and the GFP part to construct the improved part (BBa_K1139201) (Fig. 3-c).

Fig. 3-a. Our goal to create “Farmer E. coli.”

Fig. 3-b. Cucumber seed sprouts respond to cytokinin samples.

Fig. 3-d. Our phoA promoter assay result.

Fig. 3-c. Our phosphate sensor part including phoA promoter.

Fig. 3-e. Equation for the induction mechanism.

Reference: [3] Dollard et al., 2003

(The work is done by Sara and Shunta)

(The work is done by Sara)

(This assay was Carried out by Ryota)

(The work is done by Yochiku)

Our circuit had a crosstalk problem, C12HSL-LasR activates not only Plas but also Plux. In order to circumvent the crosstalk problem [1], we introduced Plux/tet and a repressor network containing cI434 and tetR.

Signal-dependent state change circuit The key part related to crosstalk, we succeeded in crosstalk circumvention by network engineering (Fig. 1-b).

Crosstalk Circumvention

Fig. 1-b Plux/tet is repressed by TetR.

Analysis of crosstalk circumvention Left figure shows the switching from expressing LacI to expressing CI. In the case which includes crosstalk circumvention circuit, switching of toggle circuit is more obvious.

Fig. 1-c. Analysis of crosstalk circumvention

(The work is done by Emma, Yuta, Hengyu)

Aizawa Foundation Tokyo Institute of Technology Found project Mr. Isao Ono Mr. Fumio Hombo

Mimic state

Attack state

E. ninja E. samurai

E. civilian

We intend to tell the public the development of synthetic biology. Through communicating with various people, we thought that we should bring the following two points into our project.

(1) Simple explanation for the importance of genetic programming (2) Application of synthetic biology that is familiar to the public

Considering these points, we created the story of “Ninja,” which is famous all over the world. By describing fascinating life of Ninja with E. coli, our project can attract a lot of people. When we design a genetic circuits of Ninja, our project will help general people understand programming of genetic network in synthetic biology.

Even if we use project "Ninja,” it is still difficult for us to appeal the technology of synthetic biology. Therefore, we started up another project "Farming.” In this project, we aim to improve efficient agriculture by using engineered bacteria for the coming food shortage. Project "Ninja" and project "Farming" are greatly promising.

We asked them to write their opinions, suggestions and questions on a label and stick it on our posters.

Flow

We introduced our ideas using posters according to visitors’ knowledge of biology.

State Switching

State Switching

An improved phosphate sensor

Fig. 2-b. The release of M13 phage particle depends on the induction of g2p expression

Fig. 1-a. Signal-dependent state change circuit

(The work is done by Yochiku and Masanori) C12 signal coming

switch!

Crosstalk!!

Reference: [1] Gray KM et al. (1994)

[µM/s]

[M]

C12

10

504

C12 +

aTc

Fluo

resc

ence

inte

nsity

(a.u

.) Part collection

Fig. 2-c.

Left picture shows the distribution of plaques.

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