iGEM 2019 — Team TokyoTech

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:TokyoTech/Notebook

[![TokyoTechproject](https://static.igem.org/mediawiki/2019/3/3b/T--TokyoTech--Projectlogo.png)](https://2019.igem.org/Team%3ATokyoTech)

* [Project](https://2019.igem.org/Team%3ATokyoTech/Description)
  + [Description](https://2019.igem.org/Team%3ATokyoTech/Description)
  + [Achievement](https://2019.igem.org/Team%3ATokyoTech/Achievement)
* [Wet Lab](https://2019.igem.org/Team%3ATokyoTech/Wet_Lab)
  + [Experiments](https://2019.igem.org/Team%3ATokyoTech/Experiments)
  + [Parts](https://2019.igem.org/Team%3ATokyoTech/Parts)
  + [Characterization](https://2019.igem.org/Team%3ATokyoTech/Characterization)
* [Dry Lab](https://2019.igem.org/Team%3ATokyoTech/Model)
  + [Modeling](https://2019.igem.org/Team%3ATokyoTech/Model)
  + [Hardware](https://2019.igem.org/Team%3ATokyoTech/Hardware)
* [Human Practices](https://2019.igem.org/Team%3ATokyoTech/Human_Practices)
  + [Specialists](https://2019.igem.org/Team%3ATokyoTech/Specialists)
  + [Education](https://2019.igem.org/Team%3ATokyoTech/Education)
  + [Team Building](https://2019.igem.org/Team%3ATokyoTech/Team_Building)
* [Team](https://2019.igem.org/Team%3ATokyoTech/Team)
  + [Members](https://2019.igem.org/Team%3ATokyoTech/Members)
  + [Judging Form](https://igem.org/2019_Judging_Form?team=TokyoTech)
  + [Collaboration](https://2019.igem.org/Team%3ATokyoTech/Collaborations)
  + [Attributions](https://2019.igem.org/Team%3ATokyoTech/Attributions)

# Notebook

Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.

### What should this page have?

* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

### Inspiration

You can see what others teams have done to organize their notes:

* [2018 Munich](https://2018.igem.org/Team%3AMunich/Notebook)
* [2014 ATOMS-Turkiye](https://2014.igem.org/Team%3AATOMS-Turkiye/Notebook)
* [2014 Tec Monterrey](https://2014.igem.org/Team%3ATec-Monterrey/ITESM14_project.html#tab_notebook)
* [2014 Kyoto](https://2014.igem.org/Team%3AKyoto/Notebook/Magnetosome_Formation#title)
* [2014 Cornell](https://2014.igem.org/Team%3ACornell/notebook)


[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:TokyoTech/Experiments

[![TokyoTechproject](https://static.igem.org/mediawiki/2019/3/3b/T--TokyoTech--Projectlogo.png)](https://2019.igem.org/Team%3ATokyoTech)

* [Project](https://2019.igem.org/Team%3ATokyoTech/Description)
  + [Description](https://2019.igem.org/Team%3ATokyoTech/Description)
  + [Achievement](https://2019.igem.org/Team%3ATokyoTech/Achievement)
* [Wet Lab](https://2019.igem.org/Team%3ATokyoTech/Wet_Lab)
  + [Experiments](https://2019.igem.org/Team%3ATokyoTech/Experiments)
  + [Parts](https://2019.igem.org/Team%3ATokyoTech/Parts)
  + [Characterization](https://2019.igem.org/Team%3ATokyoTech/Characterization)
* [Dry Lab](https://2019.igem.org/Team%3ATokyoTech/Model)
  + [Modeling](https://2019.igem.org/Team%3ATokyoTech/Model)
  + [Hardware](https://2019.igem.org/Team%3ATokyoTech/Hardware)
* [Human Practices](https://2019.igem.org/Team%3ATokyoTech/Human_Practices)
  + [Specialists](https://2019.igem.org/Team%3ATokyoTech/Specialists)
  + [Education](https://2019.igem.org/Team%3ATokyoTech/Education)
  + [Team Building](https://2019.igem.org/Team%3ATokyoTech/Team_Building)
* [Team](https://2019.igem.org/Team%3ATokyoTech/Team)
  + [Members](https://2019.igem.org/Team%3ATokyoTech/Members)
  + [Judging Form](https://igem.org/2019_Judging_Form?team=TokyoTech)
  + [Collaboration](https://2019.igem.org/Team%3ATokyoTech/Collaborations)
  + [Attributions](https://2019.igem.org/Team%3ATokyoTech/Attributions)

# Main Experiments

Turing pattern is formed by the interaction of two substances with different rate of diffusion.
One of them works as an activator and the other one works as an inhibitor.
The activator promotes its own expression and expression of the inhibitor, and the inhibitor is required to suppress the expression of the activator.

PAI1 produced by LasI gene acts as an activator, and PAI2 produced by RhlI gene acts as an inhibitor.

![](https://static.igem.org/mediawiki/2019/7/71/T--TokyoTech--%E3%83%81%E3%83%A5%E3%83%BC%E3%83%AA%E3%83%B3%E3%82%B0%E3%83%91%E3%82%BF%E3%83%BC%E3%83%B3.png)

Activator-inhibitor correlation

We will look at the specific genetic circuit function over time.

![](https://static.igem.org/mediawiki/2019/a/ac/T--TokyoTech--%E9%81%BA%E4%BC%9D%E5%AD%90%E5%9B%9E%E8%B7%AF%E5%9B%B3%E5%85%A8%E4%BD%93.png)

Gene circuit diagram

This genetic circuit begins to function when DsRed is expressed over time.When DsRed is expressed, the spot shows red fluorescence.

![](https://static.igem.org/mediawiki/2019/a/ae/T--TokyoTech--%E9%81%BA%E4%BC%9D%E5%AD%90%E5%9B%9E%E8%B7%AF%E5%9B%B3A-q.png)

Start of genetic circuit

Then, regulated by the same promoter as DsRED, LasI and RhlI produced PAI1 and PAI2 respectively. As mentioned above, they have different rate of diffusion, in which PAI2 diffuses at a higher rate compared to PAI1.

![](https://static.igem.org/mediawiki/2019/a/af/T--TokyoTech--B2.png)

Diffusion of activators and inhibitors

When diffusion occurs, PAI1, together with LasR activate itself and RhlI expression by acting on pLas-OR. This acts as an activator in the Turing pattern formation. Expression of DsRed are also promoted in the same process.

In addition, PAI2 promotes the expression of GFP and CI, while inhibiting the expression of itself and LasI/DsRed by inhibiting the function of the promoter containing the binding site. This acts as an inhibitor in the formation of Turing pattern.

![](https://static.igem.org/mediawiki/2019/6/6e/T--TokyoTech--表３.png)

Expression of DsrRed by activators and expression of GFP by inhibitors

![](https://static.igem.org/mediawiki/2019/a/a3/T--TokyoTech--PAI1.png)

Activity of PAI1 as an activator

![](https://static.igem.org/mediawiki/2019/9/98/T--TokyoTech--PAI2.png)

Working as an inhibitor of PAI2

At this point, PAI2 is also present in the range where PAI1 diffused, but because PAI1 also inhibits the binding between PAI2 and RhlR, so PAI2-induced GFP expression is suppressed in the range of diffusion by PAI1.

Also, the pattern can be manipulated by introducing other control mechanism into the promoter where RhlR / PAI2 complex binds to.

We have considered a hybrid promoter (BBa\_K3259000) that can be controlled by PAI2 and blue light, and also a hybrid promoter (BBa\_K3259001) that can be controlled by PAI2 and temperature.

---

![](https://static.igem.org/mediawiki/2019/9/96/T--TokyoTech--BBaK3259000.png)

BBa\_K3259000

With the presence of PAI2, when BBa\_K3259000 is not exposed to light, it will bind to *Escherichia coli*  -derived BluR protein, thereby suppressing gene expression. However, when it is exposed to blue light, the BluR protein will bind to the BluF protein and can no longer inhibit gene expression.

By doing so, the expression of CI and GFP becomes more active when we exposed it to blue light compared to when there is no light, and the size of red spot becomes smaller.

![](https://static.igem.org/mediawiki/2019/5/50/T--TokyoTech--mapblu.png)

---

![](https://static.igem.org/mediawiki/2019/f/f0/T--TokyoTech--BBaK3259001.png)

BBa\_K3259001

In addition, even with the presence of PAI2, the structure of promoter BBa\_K3259001 are not stable at about 30 degrees, and gene cannot be expressed. However, when it is placed at a lower temperature of about 18 degrees, the structure becomes stable and gene expression is activated.

By using this hybrid promoter, the size of red spot will become smaller at low temperatures.

![](https://static.igem.org/mediawiki/2019/1/1d/T--TokyoTech--mapcold.png)

---

In this project, we are working on ways of “controlling” patterns, but in the future we aim to create a system that can be “controlled” by environmental factors.

[![](https://static.igem.org/mediawiki/2019/3/3b/T--TokyoTech--Projectlogo.png)](https://2019.igem.org/Team%3ATokyoTech)

We are iGEM TokyoTech,

a voluntary-project group of Tokyo Institute of Technology, Japan.

Our sponsors are [here.](https://2019.igem.org/Team%3ATokyoTech/Team)

Thank you so much!!!

Copyright© iGEM TokyoTech 2019, All rights reserved

## Site Map

#### * [Home](https://2019.igem.org/Team%3ATokyoTech) * [Wet Lab](https://2019.igem.org/Team%3ATokyoTech/Wet_Lab) * [Dry Lab](https://2019.igem.org/Team%3ATokyoTech/Model) * [Human Practice](https://2019.igem.org/Team%3ATokyoTech/Human_Practices) * [Team](https://2019.igem.org/Team%3ATokyoTech/Team)

[![](https://static.igem.org/mediawiki/2019/b/bc/T--TokyoTech--nobackground-logo.png)](https://igemtokyotech2019.wixsite.com/igem-tokyotech)

#### Contact Us

##### * [Twitter](https://twitter.com/igem_tokyotech)

##### * [About Us](https://igemtokyotech2019.wixsite.com/igem-tokyotech)

[![](https://2019.igem.org/wiki/images/8/8b/HQ_page_logo.jpg)](https://2019.igem.org)

# Team:TokyoTech/Results

[![TokyoTechproject](https://static.igem.org/mediawiki/2019/3/3b/T--TokyoTech--Projectlogo.png)](https://2019.igem.org/Team%3ATokyoTech)

* [Project](https://2019.igem.org/Team%3ATokyoTech/Description)
  + [Description](https://2019.igem.org/Team%3ATokyoTech/Description)
  + [Achievement](https://2019.igem.org/Team%3ATokyoTech/Achievement)
* [Wet Lab](https://2019.igem.org/Team%3ATokyoTech/Wet_Lab)
  + [Experiments](https://2019.igem.org/Team%3ATokyoTech/Experiments)
  + [Parts](https://2019.igem.org/Team%3ATokyoTech/Parts)
  + [Characterization](https://2019.igem.org/Team%3ATokyoTech/Characterization)
* [Dry Lab](https://2019.igem.org/Team%3ATokyoTech/Model)
  + [Modeling](https://2019.igem.org/Team%3ATokyoTech/Model)
  + [Hardware](https://2019.igem.org/Team%3ATokyoTech/Hardware)
* [Human Practices](https://2019.igem.org/Team%3ATokyoTech/Human_Practices)
  + [Specialists](https://2019.igem.org/Team%3ATokyoTech/Specialists)
  + [Education](https://2019.igem.org/Team%3ATokyoTech/Education)
  + [Team Building](https://2019.igem.org/Team%3ATokyoTech/Team_Building)
* [Team](https://2019.igem.org/Team%3ATokyoTech/Team)
  + [Members](https://2019.igem.org/Team%3ATokyoTech/Members)
  + [Judging Form](https://igem.org/2019_Judging_Form?team=TokyoTech)
  + [Collaboration](https://2019.igem.org/Team%3ATokyoTech/Collaborations)
  + [Attributions](https://2019.igem.org/Team%3ATokyoTech/Attributions)

# Results

Here you can describe the results of your project and your future plans.

### What should this page contain?

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

### Describe what your results mean

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the part's Main Page on the Registry.** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

### Project Achievements

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

### Inspiration

See how other teams presented their results.

* [2014 TU Darmstadt](https://2014.igem.org/Team%3ATU_Darmstadt/Results/Pathway)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Results)
* [2014 Paris Bettencourt](https://2014.igem.org/Team%3AParis_Bettencourt/Results)