iGEM 2016 Tokyo_Tech Team has had outstanding projects every year especially in terms of experiments and modeling. This year, in order to develop further, we had hold outreach activities positively from the beginning of our project. We also had dialogues with the public and stakeholders to apply their advice to our project. Moreover, we had built this project, considering the contribution to society. Currently, synthetic biology is unfamiliar to the public, and in most cases, they cannot comprehend it easily. However, using Snow White story that everyone knows, our project is interesting and accessible. We did human practice activities to raise public interest in synthetic biology and to inspire the public to learn it with a familiar story, Snow White. What is more, our research about Toxin-Antitoxin system (TA system) and Quorum Sensing (QS) created "ACA Dwarfs" and "TA system ~Queen's Caprice~." Additionally, we are doing the experiments of E. coli mutants that secrete proteins extracellularly. It makes a giant step toward technological developments in economy in the future.

Through communicating with the public, it was shown that people tend to have an erroneous idea about genetic modification and E. coli, and they have almost no idea about synthetic biology. Additionally, there is some advice that they cannot understand the contribution to society primarily. In order to solve these problems, we created a model called 3E model to find out the contribution of this project from three aspects: education, ethics, and economy. We are aiming to make our iGEM activity have an utmost contribution to society. Additionally, we summarized how the outreach activities, the class we gave and the dialogues with experts had made changes to our project, and what we thought with advice from them and what we did in chronological order. Moreover, Snow White in Grimm's Fairy tales has been published a lot of versions so far. Although we were not able to use experiments to represent all the different versions of the story, we were able to design a genetic circuit.

We take our project into consideration from three aspects: education, ethics, and economy. From these three aspects, we can approach in various ways, thereby can grasp our project multilaterally. So far, we had worked on many activities to inform the public distinctly of E. coli, genetic modification, and synthetic biology. Once we faced some issues, we had many discussions and also tried solving the problems. Secondly, we had thought about ethics as a key point in our project and made ethics code of iGEM 2016 Tokyo_Tech Team. Further, we coordinated activities to achieve the protein production control using TA system. Although it is difficult to use commercially at present, it is a great achievement that we created the idea and technology regarded as a steppingstone. This page shows all of our human practice activities.

Overview

We consider the possibility of the protein production system is controlled by Maz system and signal transfer mechanism. To improve the performance of controlling production by Maz system, we developed a tool of increasing or decreasing the number of ACA sequence, which is the specific site cleaved by MazF homo dimmer. Maz system.

1. controlling the protein production

2. selectivity of the target mRNA

3. system of communicating with outside

Combining i. and ii., it is expected that the target protein is obtained effectively and with favorite rate. Furthermore, adding iii., we could design an automatic and advanced information-processing system. In other words, we could design an oscillatory cell-to-cell communication that one cell containing maz system receives an extrinsic signal (Maz system is controlled　by the type of the signal), then the cell produces the substance which affects other cells.

To increase the selectivity of repressing the protein production by Maz system, we develop a tool of increasing or decreasing the number of ACA sequence named “ACA Dwarfs”. This application makes our future work much more realistic than other iGEM teams (see Model page).

Furthermore, a lot of vital phenomena are oscillatory. So we could apply this oscillatory system to DDS (Drug Delivery System) or decreasing the risk of the appearance of the multidrug-resistant strains.

• Decrease the risk of the appearance the multidrug-resistant strains

  These days, it is alarmed that there appears the multidrug-resistant strains due to excessive use of pesticide. But it costs spreading the pesticide one by one. Then, we suggest using the system of controlling the protein production by Maz system: the produce of the pesticide is usually repressed by MazF, but only when the substance delivered from the target living things (for example: pheromone), MazF is contradicted by MazE and the pesticide is produced. This oscillatory model can decrease the possibility of the appearance of the multidrug-resistant strains because the pesticide is produced at intervals.

• DDS(Drug Delivery System)

  Introducing maz system to human cells and orchestrating the human original signal transduction system and Maz system, we design the mechanism that the cell secrets drug depended on the internal condition. For example, when a maz system introduced cell receives the blood glucose level increase, the cell synthesizes the insulin.

• Our Quorum Sensing and TA systems might be applicable to control the Intestinal Flora

  1.Introduction

  These days, Intestinal Flora draws a lot of attention because it will greatly affect our health or illness. If the balance of intestinal flora is lost, it will lead illness or might change your personality. It is assumed that intestinal flora consists of communication systems among various bacteria. However, no such communication systems have been found. So, it will contribute to the society to clarify the mechanism of intestinal flora and to find a way of controlling it. Our project focused on the cell-cell communication through Toxin-Antitoxin system (TA system) and Quorum Sensing (QS), and proved the initial step to explain and control the system of the intestinal symbiosis.

  2. The results of our project

  2-1. TA System Assay

  We used Maz system, which is a kind of TA system. MazF (Toxin) inhibits translation by cleaving ACA sequence in mRNA and MazE (Antitoxin) releases MazF inhibition. We showed that Maz system could regulate cell growth when mazE and mazF were alternately expressed in E. coli (see TA system assay pages).

  
  

  2-2. AHL and AmiE Assay

  We indicated that QS of Pseudomonas aeruginosa genesand V. fischeri, and amiE, which selectively degrades AHLs, could work in E. coli. Sometimes, these genes were toxic to E. coli, but it was solved by, for example, tagging ssrA or experimenting at low temperature (see AHL Reporter assay page and AmiE assay page).

  
  

  2-3. Temperature-dependent Promoters

  We indicated that temperature-dependent promoters were activated strongly at the specific temperature: gfp under Pcold, which is activated at 15℃ and under, was much more expressed at 18℃ than at 37℃; on the other hand, gfp under Pheat, which is activated at 42℃ and over, was more expressed at 37℃ than at 28℃. We can say that we can control the expression of the gene under these temperature-dependent promoters by changing temperature.

  
  

  2-4. Modeling

  We simulated our final genetic circuits containing the genes of TA system, QS and temperature-dependent promoters. The simulation showed that the circuits could perform favorably by improving some parts (see Model page and Rhl system assay page).

  3. Discussion

  In conclusion, we proved that the genes of other bacterial QS and temperature-dependent promoters worked in E. coli and that E. coli original TA system gene under expression-inducing promoters regulated the growth in WET experiments. From these results, the simulation showed that E. coli containing TA system gene and QS gene under various expression-inducing promoters communicated with each other.
  Besides, by simulating that the genetic circuits worked properly with the three types of expression-inducing promoters, we suggest that intestinal bacterial growth is controlled by nutritional condition, temperature and intestinal bacterial density. The three types of promoters were the sugar-dependent promoters (PBAD, Plac), the temperature-dependent promoters (Pcold, Pheat) and the AHL-dependent promoters (Prhl, Plux and Plas). Our project therefore helps to understand the symbiosis of intestinal flora. Furthermore, we are quite sure that we could engineer the E. coli or other bacteria which have function of regulating the symbiosis.

In promoting our project, we had dialogues with the public and experts. Based on the opinions from them, we developed our project. This led to success in creation of a well-rounded project with the connection between the public and experts, keeping from our narrow view.

This page explains the changes that some events had made to our project in chronological order.

After having dialogue with expert, we obtained the comment that TA system has the potential to be linked to the development of effective technology.

However, at present there exit problems when using a TA system to control the protein production. We cannot selectively produce only desired protein, because other proteins also would be produced at the same time.

Then our dry lab used Java to reseach into the development of ACA base sequence's number adjustable software which we call it "ACA Dwarfs" as a solution to the problem.

In this way, we integrated Human Practices to out project. However, we have done much more activities for Human Practices and show them in detail below.

Summary

We "went beyond the lab" and developed our project through human practice activities.

We have not just reflected the opinions from the dialogue with the public and experts about our project. Firstly, we investigated and discussed the issues on our own. Secondly, in the light of the application of our project, we did the experiments and modeling. Finally, we succeeded in the creation of "ACADwarfs" and "TA system～Queen's Caprice～". Furthermore, we are doing the experiments to make E. coli mutants secreting protein extracellularly. The products will probably lead to further developments of our project.

Achievements as mentioned above indicate that we have succeeded in the integrated Human Practice.

Suzukake Festival (May 14 & 15, 2016)

The Suzukake Festival, the school festival on the Suzukakedai campus, was held on May 14-15, 2016. We introduced iGEM and our projects to the visitors to let them know synthetic biology and got feedbacks.

Homecoming Day (May 21, 2016)

Homecoming Day is an annual event held at Tokyo Tech. We introduced iGEM and synthetic biology to alumni. We realized that it is hard to explain our research themes and goals to the public clearly. Therefore, we decided to make videos about E. coli and genetic modification.

Reference

The remaking of fairy tale "Snow White" by the Brothers Grimm has enjoyed a lot of versions since it was published in 1812 in the first edition of their collection Grimms" Fairy Tales. Some elements are common in any Snow White stories. However, because authors add their original elements or features to the story, it becomes unique. We chose other two versions, "Mirror Mirror" and "A Snow White Christmas" and designed their genetic circuits.