In this iGEM contest, we intend to tell the public the development of synthetic biology, especially the development of the network programming, as well as we enjoy our activity for iGEM. Tokyo_Tech 2013 assisted with 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 the importance of programming 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 aimed to program this story into E. coli, the life of ninja: battle and farming.

(go to Human Practice page)

[Fig. 1. Experiment workshop for human practice]
We assisted with an experiment workshop for high school students.

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[Fig. 2. Questionnaire survey for human practice]
We collected questionnaires from public people.

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

[Fig. 3. Our designed circuit for circumvention of the crosstalk]
We designed the circumvention of the crosstalk by network engineering.

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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” or “Attack state”. The circuit of E. ninja contains a bi-stable switch part and a signal dependent switching part. We decided to use C6-AHL and C12-AHL as the signals. The crosstalk between these two signals is well known as a significant problem in synthetic biology. To realize an accurate switching, by network engineering, we designed the circumvention of the crosstalk that occurs in bacterial cell-cell communication system (Fig. 3).

(go to State Changing page)

Our wet experiment results showed that the combination of lux/tet hybrid promoter and TetR protein circumvented the crosstalk by preventing the LasR protein from acting on LuxR-binding sequences (Fig. 4). Our mathematical model based on these results showed the circumvention of the crosstalk in the whole circuit (Fig. 5).

(go to Modeling page)

[Fig. 4. Result of our wet experiment for the circumvention of the crosstalk]
The level of GFP expression in cells where TetR is active is clearly lower than when TetR is inhibited. Even with activated LasR, lux/tet hybrid promoter is repressed by TetR precisely. This result suggest our network will circumvent the crosstalk by the activated LasR.

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[Fig. 5. Our mathematical model for the circuit of E. ninja]
The solid/dotted lines stand for the case with/without the crosstalk circumvention. The expression of LacI is repressed through the crosstalk circumvention.

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[Fig. 6 Our new part for inducible phage release]
We designed a new part for inducible phage release. Any promoter is allowed to be inserted upstream of g2p to regulate phage release.

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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 routes in synthetic biology by achieving programmed DNA messaging (Fig. 6).

(go to Shuriken page)

In the second-life story, E. ninja starts farming in a peaceful village. He can increase plant growth by synthesizing several plant hormones depending on the soil environment. We constructed an improved phosphate sensor (phoA promoter, BBa_K1139201). Also, we learned methods for quantitative analysis 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 foundations and organizations (Fig. 7).

(go to Farming page)

[Fig. 7. Our bioassay of cucumber seed sprouts]
We cultivated the sprouts in standard cytokinin sample solutions and then measured the weight of the sprouts and the concentration of chlorophyll.

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Through our project, we believe we can contribute to various fields. First, our crosstalk circumvention system gives more flexibility to design genetic circuits because of its simple network topology composed of two repressor proteins, one repressor and one hybrid promoter. Second, our inducible phage release system can make DNA messaging more complex and diverse. Moreover, for bioremediation, we can search for new M13 phage hosts by using the M13 phage we have designed. Finally, our farming project will act as a pioneering trail toward new approaches in farming. Especially, our strategy to produce plant hormones in chronological patterns in E. coli will be applied to studying the plants’ response to external plant hormones. We hope to contribute to spreading the importance and the great possibilities of synthetic biology through the public.