Team:Tokyo Tech/Medal

Achievements

Medal Criteria

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Bronze Register and attend

We registered for iGEM, had a great summer, and attended the Giant Jamboree.
Deliverables

We met all deliverables on the Requirements page.
Attribution

We created a page on our team wiki with clear attribution of each of our project.
Part

We documented 5 new BioBrick Parts (BBa_K1949100, BBa_K1949101, BBa_K1949102, BBa_K1949000, BBa_K1949001) central to our project and submitted them to the iGEM Registry .
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Silver Validated Part

We well documented experimentally validated that 4 new BioBrick Parts/Devices of our own design (BBa_K1949050, BBa_K1949052, BBa_K1949030, BBa_K1949032) and construction works as expected.
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Collaboration

We helped KAIT_Japan model their system and taught a new high school team ASIJ about modeling.
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Human Practices

We considered our project from three aspects: education, ethics, and economy. From these three aspects, we can approach in various ways, thereby we can grasp our project multilaterally.
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Gold Integrated Human Practices

We held many activities with respect to our three aspects; education, ethics and economy. For example, we gave lessons to the students at 6 different high schools (269 students in total) and held a symposium. In addition, we applied the advice and ideas got from the students and experts to our project. As a result, our Wet lab and Dry lab integrated to make "ACA Dwarfs," as well as figure out the concept of "TA system ～the Queen's Caprice～."
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Improvement / Characterization

We well improved a BioBrick Parts (BBa_R0071) and characterized 6 BioBrick Parts/Device (BBa_C0071, BBa_C0171, BBa_K1529300, Bba_K1529310, BBa_K1096002, BBa_K1096001)
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Proof of concept

We demonstrated 4 functional proof of concept of our project; New cold inducible promoter, Rhl system assay, TA system, AmiE degrades C12.
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Best Information Processing

We tried to recreate the story of “Snow White” using MazF (toxin) and MazE (antitoxin). This is the first example in which both of the toxin and the antitoxin were introduced into a single cell in iGEM competition. We experimentally confirmed that MazF inhibited translation through cleavage of mRNAs and MazE let translation “resuscitate” from the inhibition. Also, we theoretically confirmed the feasibility of desired behavior by modeling.
Our system has expanded the regulation of protein expression to the mRNA level, while the conventional regulation has mainly targeted ribosome machinery Furthermore, we succeeded to change the inhibition efficiency by designing the DNA sequence without changing the protein sequence by our new software. Therefore, we made it possible to design more various systems by combining the mazEF system and a variety of inducible promoters. This improvement will greatly expand the possibility of signal processing.


Proof of Concept

Achievement Gold

Demonstrate a functional proof of concept of your project. Your proof of concept must consist of a BioBrick device; a single BioBrick part cannot constitute a proof of concept.

1. New cold induible promoter

Here we indicated that the cold inducible promoter is a promoter that depends mainly on temperature.We performed an experiment using BBa_K1949001.


Fig. 6-3-1-1. Cold inducible promoter
the RFU of GFP / Turbidity of the samples cultivated at 18°C was higher than those cultivated at 37°C.


In this experiment, each sample was cultivated at 18°C and 37°C, after that their RFU of GFP / Turbidity was measured.

From the results, we found that the RFU of GFP / Turbidity of the samples cultivated at 18°C was higher than those cultivated at 37°C.

The above results indicate that this promoter is induced at low temperatures.

2． Rhl system assay

Here shows how we changed the rhl promoter to the one that suited our project best. We conducted an experiment using BBa_K1949060.



Fig. 6-3-2-1. Our original improved Prhl(NM) is much more sensitive to C4HSL than Prhl(WT).


A single point mutation was inserted in a wild type rhl promoter(BBa_R0071).

In the experiment, an AHL reagent was included in the reporter, and then we measured the RFU.

As a result, the mutant promoter Prhl(NM), which is stronger than a wild type promoter, was newly obtained.


Fig. 6-3-2-2. The Signal-to-Noise (SN) ratio of Prhl(NM) is higher than that of past improved Prhl(LR), and Prhl(NM) does not crosstalk to C12 unlike


iGEM 2014 team Tokyo_Tech has improved the rhl promoter. However, comparing the Prhl(NM) with the Prhl(LR), the SN ratio of Prhl(NM) was higher than that of Prhl(LR). Also, the graph shows that Prhl(LR) has crosstalk with C12 at a specific proportion. If we use it in the recreation of Snow White, and the crosstalk between the promoter and C12 will mean that the Queen eats the Poisoned Apple, which she produced herself, and dies. Therefore, this promoter cannot be used in our project.

The graph also shows that Prhl(NM) almost have no crosstalk with C12.

Based on the results above, we can say that we found a promoter with strong activity, and it almost have no crosstalk with C12, which is the most suitable one for our story.

3. TA system

Here shows the inhibition of the E. coli´s growth and translation by MazF and the resuscitation of them by MazE. We performed an experiment using BBa_K1949100 and BBa_K1949102.



Fig. 6-3-3-1. After inhibiting cell growth by MazF, it was resuscitated by expression of MazE.



Fig. 6-3-3-2. After inhibiting expression of GFP by MazF, it was resuscitated by expression of MazE．


In this experiment, MazF was expressed by arabinose, and after 2 h MazE was expressed by IPTG.

From the results, it was found that the turbidity of the sample without MazE almost did not rise at all. However, it was also found that if IPTG is added, and the MazE will be expressed, the E. coli will recover its growth.

Also, if only the MazF is working, the RFU of GFP barely rise. If MazE is induced, the RFU of GFP will rise. Read mazEF system Assay page.

As the result above, we concluded that MazF inhibits the E. coli's growth and translation. However, MazE resuscitates the growth and translation stopped by MazF.

Althrough this experiment, we indicated that the TA system functions properly.

4． Degredation of C12 by AmiE

Here it is indicated that AmiE degrades AHL selectively. We performed an experiment using BBa_K1949052.



Fig. 6-3-4-1. AmiE degrades C12   Fig. 6-3-4-2. AmiE barely degrades C４
C12 is greatly degraded. On the other hand, the C4 is barely degraded by AmiE.


In this experiment, it was examined whether AHLs would be degraded after the addition of 3OC12HSL(C12) and C4HSL(C4) molecules into a culture of E. coli that expresses AmiE.
From the results, it was found that the C12 added into a culture of AmiE expressing E. coli is greatly degraded. On the other hand, the C4 added into this culture, is barely degraded.

From the above result, we showed that AmiE selectively degraded AHL molecules.

Human Practices

Achivement Silver

Education

  • School-visits

    In order to provide more people in general with a more accurate information about E. coli, gene reconfiguration, and synthetic biology, we planed to conduct some School-visits. Finally, we have managed to give the classes to 269 middle and high school students of 6 schools. Furthermore, these activities were good opportunities for us to know how our project impacted on the general public. The details are organized for each school so that anyone can do these kind of lessons in the future. Please feel free to use these as reference.

  • Video

    Because the public could not understand our activities well, we made intelligible videos to improve this situation. The videos are delivering over YouTube.

  • Card Game

    We made the card game for the public to understand the basis of our project, TA system and Quorum Sensing.

  • An Expert

    We noticed that ways of manifesting and expressing our project are important when we explain our project to the public. An expert gave us the advice that we should insert something extremely impressive for the public to summarize our project in accordance to audience’s satisfaction.

Ethics

  • Symposium

    We hosted a symposium providing an opportunity for students to think about synthetic biology.

  • Code of ethics

    Based on the symposium, we have developed our original code of ethics.

Economy

  • Dialogue with an expert

    Through developing ideas aroused from dialogue with an expert, we have created “TA system ~Queen’s Caprice~” and “ACADwarfs.” Furthermore, getting a hint from it, we have made experiments of E. coli mutants secreting proteins extracellularly. In order to realize more efficient control of protein synthesis, this is an approach of our human practice activities from a perspective of economy.

Snow White Versions

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 as follow.