iGEM 2017 — Team Botchan_Lab_Tokyo

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# Team:Botchan Lab Tokyo/Notebook

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

PROJECT

[RESULTS](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Project)
[LABORATORY NOTEBOOK](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Notebook)
[MODELING](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Model)

LAB

[TEAM](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Team)
[ATTRIBUTIONS](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Attributions)
[INTERLAB](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/InterLab)

HUMAN PRACTICE

[SILVER](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/HP/Silver)
[GOLD](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/HP/Gold_Integrated)
[COLLABOLATIONS](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Collaborations)

# Notebook

![](https://static.igem.org/mediawiki/2017/d/d4/T--Botchan_Lab_Tokyo--_Results_marker_size.jpg)

We used the same marker for the result of electrophoresis.

Jun 29
• LD medium preparation

July 3
• Nitrogen-free medium A and B preparation

July 5
• Nitrogen-free medium C and D preparation
• glutamate solution and NH4Cl solution preparation

July 20
• *ATCC15970* is planted in a LD liquid medium

July 21
• *ATCC15970* is planted in a Nitrogen-free medium from LD liquid medium
• Filling of nitrogen

July 24
• Indophenol (72h culture)
"We forgot to take a picture."

July 27
• PCR of *ATCC15970* and pUC19

July 28
• Electrophoresis of PCR product

![](https://static.igem.org/mediawiki/2017/d/db/T--Botchan_Lab_Tokyo--note_ab.png)
Figure 1 Electrophoresis of PCR product

July 31
• In Fusion Cloning of *NifBHDKENXhesA* and pUC

August 1
• Transformation

![](https://static.igem.org/mediawiki/2017/9/9d/T--Botchan_Lab_Tokyo--note_ac.png)
Figure 2 Transformation colony

August 3
• PCR of *NBRC 105701* and *glu DH,phe DH*

August 8
• Insert check PCR of *NifBHDKENXhesA*
• LB+Km medium preparation

August 9
• Electrophoresis of Insert check PCR product and *NBRC 105701*,*glu DH,phe DH* PCR product

![](https://static.igem.org/mediawiki/2017/2/2e/T--Botchan_Lab_Tokyo--note_ad.png)
Figure 3 Electrophoresis of Insert check PCR product and *NBRC 105701*,*glu DH,phe DH* PCR product

August 22
• *ATCC15970* is planted in a LB+Amp medium from A

August 23
• Plasmid extraction of transgenic organism
• Electrophoresis of Plasmid extraction product

![](https://static.igem.org/mediawiki/2017/b/b8/T--Botchan_Lab_Tokyo--note_ae.png)
Figure 4 Electrophoresis of Plasmid extraction product

August 24
• Indophenol

![](https://static.igem.org/mediawiki/2017/d/df/T--Botchan_Lab_Tokyo--note_af.jpg)
Figure 5 Indophenol

August 25
• Indophenol

![](https://static.igem.org/mediawiki/2017/1/13/T--Botchan_Lab_Tokyo--note_ag.jpg)

![](https://static.igem.org/mediawiki/2017/1/17/T--Botchan_Lab_Tokyo--note_ah.jpg)
Figure 6,7 Indophenol

August 28
• Transgenic organism is planted in a LD liquid medium

August 29
• Plasmid extraction of transgenic organism
• Restriction enzyme treatment of restriction enzyme treatment

August 30
• Ethanol precipitation of restriction enzyme treatment product
• Electrophoresis of restriction enzyme treatment product and no restriction enzyme treatment product

![](https://static.igem.org/mediawiki/2017/8/88/T--Botchan_Lab_Tokyo--note_ai.jpg)
Figure 8 Electrophoresis of restriction enzyme treatment product and no restriction enzyme treatment product

August 31
• Plasmid extraction of transgenic organism
• Electrophoresis of Plasmid extraction product

![](https://static.igem.org/mediawiki/2017/5/57/T--Botchan_Lab_Tokyo--note_aj.jpg)
Figure 9 Electrophoresis of Plasmid extraction product

• Restriction enzyme treatment of restriction enzyme treatment
• Electrophoresis of restriction enzyme treatment product and no restriction enzyme treatment product

![](https://static.igem.org/mediawiki/2017/a/a0/T--Botchan_Lab_Tokyo--note_ak.jpg)
Figure 10 Electrophoresis of restriction enzyme treatment product and no restriction enzyme treatment product

September 5
• Insert check PCR of *NifBHDKENXhesA*

September 6
• Electrophoresis of PCR product
• PCR of Plasmid extraction product

![](https://static.igem.org/mediawiki/2017/0/06/T--Botchan_Lab_Tokyo--note_al.jpg)
Figure 11 Electrophoresis of PCR product

September 7
• Electrophoresis of PCR product
• PCR of pUC19

![](https://static.igem.org/mediawiki/2017/e/eb/T--Botchan_Lab_Tokyo--note_am.jpg)
Figure 12 Electrophoresis of PCR product

September 8
• Electrophoresis of pUC19 PCR product

![](https://static.igem.org/mediawiki/2017/0/03/T--Botchan_Lab_Tokyo--note_an.jpg)
Figure 13 Electrophoresis of PCR product

September 18
• *ATCC15970* is planted in a LD liquid medium

September 19
• *ATCC15970* is planted in a Nitrogen-free medium from LD liquid medium
• Filling of nitrogen

September 20
• Indophenol (24h culture)

September 21
• PCR of *pHY300PKL,pUC19* and *ATCC15970*

September 22
• Electrophoresis of PCR product

![](https://static.igem.org/mediawiki/2017/d/d6/T--Botchan_Lab_Tokyo--note_ao.jpg)
Figure 14 Electrophoresis of PCR product

• Indophenol (72h culture)

![](https://static.igem.org/mediawiki/2017/b/b3/T--Botchan_Lab_Tokyo--note_ap.jpg)
Figure 15 Indophenol (72h culture)

September 25
• Purification of PCR product
• Electrophoresis of purification product

![](https://static.igem.org/mediawiki/2017/4/40/T--Botchan_Lab_Tokyo--note_aq.jpg)
Figure 16 Electrophoresis of PCR product

September 26
• Electrophoresis of purification product
• PCR of *ATCC15970*

![](https://static.igem.org/mediawiki/2017/9/92/T--Botchan_Lab_Tokyo--note_ar.jpg)
Figure 17 Electrophoresis of PCR product

September 27
• Electrophoresis of PCR product

![](https://static.igem.org/mediawiki/2017/8/84/T--Botchan_Lab_Tokyo--note_as.jpg)
Figure 18 Electrophoresis of PCR product

• Purification of PCR product
• Electrophoresis of purification product

![](https://static.igem.org/mediawiki/2017/b/b2/T--Botchan_Lab_Tokyo--note_at.jpg)
Figure 19 Electrophoresis of purification product

September 28
• Transformation

October 2
• Transformation
• *ATCC15970* is planted in a LD liquid medium

October 3
• *ATCC15970* is planted in a Nitrogen-free medium from LD liquid medium
• Filling of nitrogen

October 4
• Indophenol (24h culture)

![](https://static.igem.org/mediawiki/2017/2/23/T--Botchan_Lab_Tokyo--note_au.jpg)
Figure 20 Indophenol (24h culture)

October 5
• Indophenol (48h culture)

![](https://static.igem.org/mediawiki/2017/8/89/T--Botchan_Lab_Tokyo--note_av.jpg)
Figure 21 Indophenol (48h culture)

• In Fusion Cloning of *NifBHDKENXhesA* and pUC/ *NifBHDKENXhesA* and *pHY*
• Transformation

![](https://static.igem.org/mediawiki/2017/5/5c/T--Botchan_Lab_Tokyo--note_aw.jpg)

![](https://static.igem.org/mediawiki/2017/b/b3/T--Botchan_Lab_Tokyo--note_ax.jpg)
Figure 22,23 Transformation colony

October 6
• Indophenol (72h culture)

![](https://static.igem.org/mediawiki/2017/c/c1/T--Botchan_Lab_Tokyo--note_ay.jpg)
Figure 24 Indophenol (72h culture)

• Transformation

![](https://static.igem.org/mediawiki/2017/2/2b/T--Botchan_Lab_Tokyo--note_az.jpg)

![](https://static.igem.org/mediawiki/2017/2/2a/T--Botchan_Lab_Tokyo--note_ba.jpg)
Figure 25,26 Transformation colony

October 9
• Preculture of Transformation sample

October 10
• Plasmid extraction
• Electrophoresis of plasmid extraction

![](https://static.igem.org/mediawiki/2017/8/85/T--Botchan_Lab_Tokyo--note_bb.jpg)
Figure 27 Electrophoresis of plasmid extraction

October 12
• Insert check PCR of *NifBHDKENXhesA*
• Electrophoresis of PCR product

![](https://static.igem.org/mediawiki/2017/8/88/T--Botchan_Lab_Tokyo--note_bc.jpg)
Figure 28 Electrophoresis of PCR product

October 13
• Transformation

October 16
• PCR of *NifVSU*
• Electrophoresis of PCR product
• Plasmid extraction

![](https://static.igem.org/mediawiki/2017/7/73/T--Botchan_Lab_Tokyo--note_bd.jpg)
Figure 29 Electrophoresis of PCR product

![](https://static.igem.org/mediawiki/2017/0/02/T--Botchan_Lab_Tokyo--note_be.jpg)
Figure 30 Electrophoresis of Plasmid extraction product

October 17
• Restriction enzyme treatment with Sac1,Xma1
• *ATCC15970* and Transformants is planted in a LD liquid medium

October 18
• Ligation of *NifVSU* and pMW218
• Transformation

![](https://static.igem.org/mediawiki/2017/1/13/T--Botchan_Lab_Tokyo--note_bf.jpg)
Figure 31 Transformation colony

• *ATCC15970* and Transformants is planted in a Nitrogen-free medium from LD liquid medium
• Filling of nitrogen

October 19
• Indophenol (24h culture)

![](https://static.igem.org/mediawiki/2017/3/3e/T--Botchan_Lab_Tokyo--note_bg.jpg)
Figure 32 Indophenol (24h culture)

• Preculture of Transformation sample

October 20
• Plasmid extraction
• PCR of transformations colony
• Electrophoresis of PCR product

![](https://static.igem.org/mediawiki/2017/9/9f/T--Botchan_Lab_Tokyo--note_bh.jpg)
Figure 33 Electrophoresis of PCR product

• Indophenol (48h culture)

![](https://static.igem.org/mediawiki/2017/0/07/T--Botchan_Lab_Tokyo--note_bi.jpg)
Figure 34 Indophenol (48h culture)

October 21
• Electrophoresis of Plasmid extraction product

![](https://static.igem.org/mediawiki/2017/e/e4/T--Botchan_Lab_Tokyo--note_bj.jpg)
Figure 35 Electrophoresis of Plasmid extraction product

October 24
• *ATCC15970* and Transformants is planted in a LD liquid medium

October 25
• *ATCC15970* and Transformants is planted in a Nitrogen-free medium from LD liquid medium
• Filling of nitrogen

October 26
• Indophenol (24h culture)

![](https://static.igem.org/mediawiki/2017/8/80/T--Botchan_Lab_Tokyo--note_bk.jpg)
Figure 36 Indophenol (24h culture)

• *ATCC15970* and Transformants is planted in a LD liquid medium
• PCR of transformations colony

October 27
• Electrophoresis of PCR product

![](https://static.igem.org/mediawiki/2017/e/e1/T--Botchan_Lab_Tokyo--note_bl.jpg)

![](https://static.igem.org/mediawiki/2017/8/84/T--Botchan_Lab_Tokyo--note_bm.jpg)
Figure 37,38 Electrophoresis of PCR product

• PCR of *pheDH* and *gluDH*
• Electrophoresis of PCR product

![](https://static.igem.org/mediawiki/2017/d/d9/T--Botchan_Lab_Tokyo--note_bn.jpg)
Figure 39 Electrophoresis of PCR product

• *ATCC15970* and Transformants is planted in a Nitrogen-free medium from LD liquid medium
• Filling of nitrogen
• Indophenol (48h culture)

![](https://static.igem.org/mediawiki/2017/7/73/T--Botchan_Lab_Tokyo--note_bo.jpg)
Figure 40 Indophenol (48h culture)

October 30
• Indophenol (72h culture)

![](https://static.igem.org/mediawiki/2017/0/09/T--Botchan_Lab_Tokyo--note_bp.jpg)
Figure 41 Indophenol (72h culture) (without LD medium version)

October 31
• Preculture of Transformation(*NifVSU*) sample

November 1
• Plasmid extraction
• Electrophoresis of Plasmid extraction product

![](https://static.igem.org/mediawiki/2017/8/80/T--Botchan_Lab_Tokyo--note_bq.jpg)
Figure 42 Electrophoresis of Plasmid extraction product

## Sponsor

[![](https://static.igem.org/mediawiki/2017/1/1d/T--Botchan_Lab_Tokyo--botchanlab_sponser4.jpg)](https://www.promega.jp/)
[![](https://static.igem.org/mediawiki/2017/8/81/T--Botchan_Lab_Tokyo--footer_logo5m.png)](http://metagen.co.jp/)
[![](https://static.igem.org/mediawiki/2017/5/5b/T--Botchan_Lab_Tokyo--botchanlab_sponser1.jpg)](http://sg.idtdna.com/site)

[![](https://static.igem.org/mediawiki/2017/9/9c/T--Botchan_Lab_Tokyo--botchanlab_sponser2.jpg)](https://www.cosmobio.com/)
[![](https://static.igem.org/mediawiki/2017/8/8f/T--Botchan_Lab_Tokyo--botchanlab_sponser3.jpg)](https://lne.st/)

### Contact

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# Team:Botchan Lab Tokyo/Experiments

[![](http://placehold.it/350x150)](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo)

MENU

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

TEAM

[Team](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Team)
[Collaborations](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Collaborations)

PROJECT

[Description](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Description)
[Design](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Design)
[Experiments](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Experiments)
[Notebook](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Notebook)
[InterLab](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/InterLab)
[Contribution](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Contribution)
[Model](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Model)
[Results](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Results)
[Demonstrate](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Demonstrate)
[Improve](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Improve)
[Attributions](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Attributions)

PARTS

[Parts](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Parts)
[Basic Parts](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Basic_Part)
[Composite Parts](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Composite_Part)
[Part Collection](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Part_Collection)

[SAFETY](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Safety)

HUMAN PRACTICES

[Silver HP](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/HP/Silver)
[Integrated and Gold](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/HP/Gold_Integrated)
[Public Engagement](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Engagement)

AWARDS

[Applied Design](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Applied_Design)
[Entrepreneurship](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Entrepreneurship)
[Hardware](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Hardware)
[Measurement](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Measurement)
[Model](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Model)
[Plant](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Plant)
[Software](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Software)

[JUDGING FORM](https://igem.org/2017_Judging_Form?team=Botchan_Lab_Tokyo)

# Botchan\_Lab\_Tokyo

# Experiments

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

Please remember to put all characterization and measurement data for your parts on the corresponding Registry part pages.

##### What should this page contain?

* Protocols
* Experiments
* Documentation of the development of your project

##### Inspiration

* [2014 Colombia](https://2014.igem.org/Team%3AColombia/Protocols)
* [2014 Imperial](https://2014.igem.org/Team%3AImperial/Protocols)
* [2014 Caltech](https://2014.igem.org/Team%3ACaltech/Project/Experiments)

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

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

# Team:Botchan Lab Tokyo/Results

[![](http://placehold.it/350x150)](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo)

MENU

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

TEAM

[Team](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Team)
[Collaborations](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Collaborations)

PROJECT

[Description](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Description)
[Design](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Design)
[Experiments](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Experiments)
[Notebook](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Notebook)
[InterLab](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/InterLab)
[Contribution](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Contribution)
[Model](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Model)
[Results](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Results)
[Demonstrate](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Demonstrate)
[Improve](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Improve)
[Attributions](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Attributions)

PARTS

[Parts](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Parts)
[Basic Parts](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Basic_Part)
[Composite Parts](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Composite_Part)
[Part Collection](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Part_Collection)

[SAFETY](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Safety)

HUMAN PRACTICES

[Silver HP](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/HP/Silver)
[Integrated and Gold](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/HP/Gold_Integrated)
[Public Engagement](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Engagement)

AWARDS

[Applied Design](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Applied_Design)
[Entrepreneurship](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Entrepreneurship)
[Hardware](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Hardware)
[Measurement](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Measurement)
[Model](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Model)
[Plant](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Plant)
[Software](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Software)

[JUDGING FORM](https://igem.org/2017_Judging_Form?team=Botchan_Lab_Tokyo)

# Botchan\_Lab\_Tokyo

# 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)

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