We intend to send a message to society so as to let them know the development of synthetic biology, especially the development of the network programming, as well as we enjoy our activity for iGEM.
Tokyo_tech2013 joined an “experiment workshop” for high school students and collected questionnaires from public people as human practice. (Fig.1) Now we know that an interesting story makes general people easily understand importance of programming of genetic circuits in synthetic biology. (Fig.2) To respond to the public's expectations further, we also look to address a farming issue. Thus we made this story, the life of the ninja: battle and farming.

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[Fig1. Human Practice experiment]
We did experiment class for high school students.

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[Fig2. Human Practice questionnaire]
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Ninja is a Japan’s ancient spy-warrior. Usually Ninja disguises himself as an ordinary civilian in public. Once he detects samurai who is the assassination target, he immediately gets ready for battle. He defeats samurai with shuriken, throwing knives.

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[Fig3. E.ninja genetic circuit]
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In our programing of artificial genetic circuit, E. ninja heads the cast. In response to E. civilian signal or E. samurai signal, E. ninja changes its state: “mimic state” and “attack state”. The circuit of E. ninja contains a bi-stable switch and a signal dependent switching part. We decided to use C6-AHL and C12-AHL as signals. The crosstalk between the two signals in synthetic biology, is well known as a big problem to be addressed. To realize accurate switching, by network engineering, we designed circumvention of the crosstalk that occurs in bacterial cell-cell communication system. (fig.3)

<a href="">(go to State Changing page)</a>

Our wet experiments shows combination of lux/tet hybrid promoter and TetR prohibited the cross talk of activated LasR protein to luxR-binding sequences (fig4.) Mathematical modeling including results from our wet experiments shows the circumvention in the whole circuit(Fig.5) (please compare green solid and dotted lines.).

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[Fig4. Crosstalk assay]
This graph shows that Plux/tet hybrid promoter can suppress the crosstalk.

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[Fig5. Modeling]
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[Fig6. M13 phage plaque]
We confirmed inducible M13 release by plaque assay.

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In addition, E. ninja releases M13 phage, which corresponds to shuriken, when it receives E. samurai signal. The inducible phage release will open new synthetic biology by programmed DNA messaging. (fig.6)

<a href="">(go to Shuriken page)</a>

In the second-life story, E. ninja starts farming in a peaceful village. It can synthesize plant hormone efficiently, depending on the soil environment. We constructed a new phosphate sensor (phoA promoter). Also, we learned methods for quantitative analysis for activity of cytokinin, a plant hormone, through a bioassay of cucumber seed sprouts. Towards further consideration of farming with microbes, we have also continued the human practice investigation through some interviews with Science Foundation or Organizations spreading the science. (fig.7)

<a href="">(go to Farming page)</a>

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[Fig7. Bioassay of plant]
We learned methods for quantitative analysis for cytokinin, a plant hormone, through a bioassay of cucumber seed sprouts.

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