iGEM 2017 — Team Kobe

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# Team:Kobe/Notebook

* ![](https://static.igem.org/mediawiki/2017/c/c0/T--Kobe--teamlogo.jpg)
* MENU

* * [HOME](https://2017.igem.org/Team%3AKobe)

  PROJECT

  + [Background](https://2017.igem.org/Team%3AKobe/Background)
  + [Overview](https://2017.igem.org/Team%3AKobe/Overview)
  + [Design](https://2017.igem.org/Team%3AKobe/Design)
  + [Parts](https://2017.igem.org/Team%3AKobe/Parts)
  + [Experiments](https://2017.igem.org/Team%3AKobe/Experiments)
  + [Results & Discussions](https://2017.igem.org/Team%3AKobe/RandD)
  + [Notebook](https://2017.igem.org/Team%3AKobe/Notebook)
* [SAFETY](https://2017.igem.org/Team%3AKobe/Safety)

HUMAN PRACTICE

+ [Integrated Human Practice](https://2017.igem.org/Team%3AKobe/HP/Gold_Integrated)
+ [Education and Public Engagement](https://2017.igem.org/Team%3AKobe/HP/Silver)

* [INTERLAB](https://2017.igem.org/Team%3AKobe/InterLab)
* ABOUT US

  + [Meet the Team](https://2017.igem.org/Team%3AKobe/Team)
  + [Attributions](https://2017.igem.org/Team%3AKobe/Attributions)
  + [Collaborations](https://2017.igem.org/Team%3AKobe/Collaborations)
  + [Acknowledgements and Sponsors](https://2017.igem.org/Team%3AKobe/Acknowledgement)

# Notebook

### ・protocol

Click
[here](https://static.igem.org/mediawiki/2017/c/cc/T--Kobe--notebook1.pdf)
for our Protocols

### ・journal

Click
[here](https://static.igem.org/mediawiki/2017/0/02/T--Kobe--notebook2.pdf)
for our Journal

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[![](https://static.igem.org/mediawiki/2017/8/8b/HQ_page_logo.jpg)](https://2017.igem.org)

# Team:Kobe/Experiments

* ![](https://static.igem.org/mediawiki/2017/c/c0/T--Kobe--teamlogo.jpg)
* MENU

* * [HOME](https://2017.igem.org/Team%3AKobe)

  PROJECT

  + [Background](https://2017.igem.org/Team%3AKobe/Background)
  + [Overview](https://2017.igem.org/Team%3AKobe/Overview)
  + [Design](https://2017.igem.org/Team%3AKobe/Design)
  + [Parts](https://2017.igem.org/Team%3AKobe/Parts)
  + [Experiments](https://2017.igem.org/Team%3AKobe/Experiments)
  + [Results & Discussions](https://2017.igem.org/Team%3AKobe/RandD)
  + [Notebook](https://2017.igem.org/Team%3AKobe/Notebook)
* [SAFETY](https://2017.igem.org/Team%3AKobe/Safety)

HUMAN PRACTICE

+ [Integrated Human Practice](https://2017.igem.org/Team%3AKobe/HP/Gold_Integrated)
+ [Education and Public Engagement](https://2017.igem.org/Team%3AKobe/HP/Silver)

* [INTERLAB](https://2017.igem.org/Team%3AKobe/InterLab)
* ABOUT US

  + [Meet the Team](https://2017.igem.org/Team%3AKobe/Team)
  + [Attributions](https://2017.igem.org/Team%3AKobe/Attributions)
  + [Collaborations](https://2017.igem.org/Team%3AKobe/Collaborations)
  + [Acknowledgements and Sponsors](https://2017.igem.org/Team%3AKobe/Acknowledgement)

# Experiments

In this page, we are going to describe the overview of the research and experiments during the iGEM activity. Please go through our Notebook for the detailed protocols.

Our project consists of 6 main steps.

1. [Growth test](#a7)
2. [RNA extraction](#a1)
3. [RNA-seq](#a2)
4. [RT-qPCR](#a3)
5. [Transformation](#a4)
6. [Beta-galactosidase assay](#a5)

### 1. Growth test

As stated in overview, our project depends on the theanine responsive gene the bacteria have. Roughly predicted, if bacteria can assimilate L-theanine, at least one of their genes related to the assimilation of L-theanine must be strongly induced in the presence of L-theanine.
Based on this assumption, we carried out a simple screening test to see whether the bacteria could use L-theanine as a sole nitrogen source.

We arranged 2 strains of *E. coli* and 3 strains of *B. subtilis* and cultivated them on the derivative of M9 minimum agar media (supplemented with L-theanine as a sole nitrogen source).

As a result, **B. subtilis** 168 and **B. subtilis** NCIB 3610 could grow using L-theanine as sole nitrogen source. Finally, we decided to analyze the genes expression of **B. subtilis** NCIB 3610 and identified the genes which were active only in the presence of L-theanine. (Please visit our
[result page](https://2017.igem.org/Team%3AKobe/RandD)
for more information)

### 2. RNA extraction

In order to identify the "theanine responsive genes", we compared the whole gene expression in *B. subtilis* NCIB 3610 depending on different nitrogen sources (L-theanine, glutamate, water, L-theanine+glutamate). Before we extracted RNA samples from the cells, we worked at developing the medium for RNA sampling. This medium was synthesized based on M9 minimum media with a little amount of yeast extract as a nitrogen source. After setting the condition, we cultivated the bacteria in each media and extracted RNA from each sample.

#### Protocols

1. 500ml of following solution was prepared in 500 ml of Erlenmeyer flask.
   (10×M9、0.5％glucose, 0.0001M CaCl
   2, 0.002M MgSO
   4, Trace element, 0.05g /L NH
   4Cl, 0.02% Yeast Extract, water)
   Cells were inoculated at OD
   600 of 0.02. and grown at 37℃, 180 rpm.
2. When the OD reached 0.15, L-theanine (final concentration 18.7mM), glutamate (final concentration 1.1 mM), L-theanine (18.7 mM)+glutamate (1.1 mM) ,and water were added to each flask.
3. Take the medium as a reference. When the OD
   600 reached 0.3, 1 ml of culture was transferred to a fresh Eppendorf tube.
4. The cells were collected by centrifugation for 10 min (4℃, 6000 rpm). mRNA was extracted according to the protocols as described in the Protocol page.

### 3. RNA－seq

#### What is RNA-seq?

RNA-seq is a powerful technique to analyze the gene expression in different conditions (different tissues or different stage of developments).

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# Team:Kobe/Results

* ![](https://static.igem.org/mediawiki/2017/c/c0/T--Kobe--teamlogo.jpg)
* MENU

* * [HOME](https://2017.igem.org/Team%3AKobe)

  PROJECT

  + [Background](https://2017.igem.org/Team%3AKobe/Background)
  + [Overview](https://2017.igem.org/Team%3AKobe/Overview)
  + [Design](https://2017.igem.org/Team%3AKobe/Design)
  + [Parts](https://2017.igem.org/Team%3AKobe/Parts)
  + [Experiments](https://2017.igem.org/Team%3AKobe/Experiments)
  + [Results & Discussions](https://2017.igem.org/Team%3AKobe/RandD)
  + [Notebook](https://2017.igem.org/Team%3AKobe/Notebook)
* [SAFETY](https://2017.igem.org/Team%3AKobe/Safety)

HUMAN PRACTICE

+ [Integrated Human Practice](https://2017.igem.org/Team%3AKobe/HP/Gold_Integrated)
+ [Education and Public Engagement](https://2017.igem.org/Team%3AKobe/HP/Silver)

* [INTERLAB](https://2017.igem.org/Team%3AKobe/InterLab)
* ABOUT US

  + [Meet the Team](https://2017.igem.org/Team%3AKobe/Team)
  + [Attributions](https://2017.igem.org/Team%3AKobe/Attributions)
  + [Collaborations](https://2017.igem.org/Team%3AKobe/Collaborations)
  + [Acknowledgements and Sponsors](https://2017.igem.org/Team%3AKobe/Acknowledgement)

# 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.

##### You should also 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 be on part pages in the Registry.
* 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)

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