iGEM 2020 — Team Botchan_Lab_Tokyo

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Botchan Lab Tokyo/Notebook

* [HOME](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo)
* Project
  + [Description](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Description)
  + [Contribution](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Contribution)
  + [Engineering Success](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Engineering)
  + [Proposed Implementation](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Implementation)
  + [Hardware](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Hardware)
* [Safety](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Safety)
* Human&Practice
  + [collaboration](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Collaborations)
  + [Integrated Human&Practice](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Human_Practices)
* [Partnership](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Partnership)
* [Science Communication](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Education)
* Team
  + [member](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/member)
  + [Attributions](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Attributions)
  + [contact](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/contact)

# 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://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Botchan Lab Tokyo/Engineering

* [HOME](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo)
* Project
  + [Description](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Description)
  + [Contribution](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Contribution)
  + [Engineering Success](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Engineering)
  + [Proposed Implementation](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Implementation)
  + [Hardware](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Hardware)
* [Safety](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Safety)
* Human&Practice
  + [collaboration](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Collaborations)
  + [Integrated Human&Practice](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Human_Practices)
* [Partnership](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Partnership)
* [Science Communication](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Education)
* Team
  + [member](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/member)
  + [Attributions](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Attributions)
  + [contact](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/contact)

# Project

## Engineering success

Although we could not get into laboratory and conduct experiments, we were ready to conduct experiments.

##### Extraction of nicotine

We will follow the experimental procedure as we showed on the extraction of nicotine page. ([extraction of nicotine](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Description#jump-nico)) We contacted cigarette manufacturers and volunteer organizations that collect cigarette butts and negotiated with them to get their cigarette waste.As a result, we were able to learn about the tobacco farmer's waste and made a commitment to provide us with the cigarette butts they picked up. we were lucky to get samples of tobacco waste as shown on figure 1. All other materials are ready at laboratory.

![](https://static.igem.org/mediawiki/2020/4/44/T--Botchan_Lab_Tokyo--SUIGARAnoGAZOU.png)

Figure1. image of the tobacco waste.

##### Plan A (*E. coli* as chassis)

For the overview of this plan, please check the description([Converting nicotine into 5-aminolevulinic acid](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Description#jump-connico)) First, we will insert genes form metabolic pathway of *P. putida* S16 and check if they function as previously reported. We codon optimized the genes using IDT codon optimization tool. We will ask IDT to synthesis our DNA fragments including promoter([Part:BBa\_J23100](http://parts.igem.org/Part%3ABBa_J23100)), RBS([BBa\_B0034](http://parts.igem.org/Part%3ABBa_B0034)) and terminator([BBa\_B0015](http://parts.igem.org/Part%3ABBa_B0015)). We will insert the genes to [pSB1C3 vector](http://parts.igem.org/Part%3ApSB1C3) by Gibson assembly. You can see plasmid maps of them on figure 2. After confirming the transformation of plasmid DNA to *E. coli* by restriction enzymes, we will add the cells to water containing substrates and check if they degrade them by thin layer chromatography. After this is checked, we will introduce plasmid containing *luxI*, *luxR*, lux promoter, and *gfp*.(*lux*-*gfp*-pSB1C3 on figure2) This is for checking the timing of the expression of genes under lux promoter. By assembling all together, we will get the *E. coli* we want.

Figure 2. Links to the plasmid maps.

| Name of the plasmid | Link |
| *nicA2* expression device | <https://benchling.com/s/seq-xsEECePdAspGs5OF7iPY> |
| *pnao* expression device | <https://benchling.com/s/seq-05GEiNs82c0DrFPus4VN> |
| *sapd* expression device | <https://benchling.com/s/seq-e0fK86luSbKmeGxjoWmt> |
| *spmABC* expression device | <https://benchling.com/s/seq-DDvHTheksQ4L1TG8oy7G> |
| *hspB* expression device | <https://benchling.com/s/seq-mKBtdNJpU0GR42sT9zgt> |
| *lux*-*gfp*-pSB1C3 | <https://benchling.com/s/seq-MkBOYJrJjj2g81NE8S8D> |

##### PlanB(*P. putida* S16 as chassis)

For the overview of this plan, please check the description. ([Using *Pseudomonas putida* S16 as a chassis](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Description#jump-usinge)) First we will make a *hpo* deletion mutant. This is done by introducing *hpo* plasmid and homologous recombination. (figure 3.) After incubating *P. putida* on kanamycin plasmid and checking deletion by PCR, we will transform plasmid containing *lux*, *luxR*, lux promoter, *tetR*, tet promoter, and *gfp* by triparental mating. This is for checking the timing of the expression of genes under lux system and tet promotor. Finally, changing the *gfp* to *hpo*, we will complement *hpo*.

Figure 3. Links to the plasmid maps

| Name of the plasmid | Link |
| pK18mob-*hpo*-knockout-plasmid | <https://benchling.com/s/seq-LVzBsD02LI1jKUFznixF> |
| *lux*-*tet*-pK18mob | <https://benchling.com/s/seq-xpfbH4Cg0hDm1L9T54mh> |
| *hpo* complement device | <https://benchling.com/s/seq-JcAzK3pPjkcB1geGuAO8> |

##### Converting to 5-aminolevulinic acid.

For the overview of this, please check the description. ([Converting 2,5-hydroxypyridine to 5-aminolevulinic acid](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Description#jump-con25)) We will conduct experiments as shown on the page.

[![](https://static.igem.org/mediawiki/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Botchan Lab Tokyo/Experiments

* [HOME](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo)
* Project
  + [Description](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Description)
  + [Contribution](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Contribution)
  + [Engineering Success](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Engineering)
  + [Proposed Implementation](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Implementation)
  + [Hardware](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Hardware)
* [Safety](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Safety)
* Human&Practice
  + [collaboration](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Collaborations)
  + [Integrated Human&Practice](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Human_Practices)
* [Partnership](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Partnership)
* [Science Communication](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Education)
* Team
  + [member](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/member)
  + [Attributions](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Attributions)
  + [contact](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/contact)

# 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.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).

### What should this page contain?

* Protocols
* Experiments

### 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/2020/8/8b/HQ_page_logo.jpg)](https://2020.igem.org)

# Team:Botchan Lab Tokyo/Results

* [HOME](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo)
* Project
  + [Description](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Description)
  + [Contribution](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Contribution)
  + [Engineering Success](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Engineering)
  + [Proposed Implementation](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Implementation)
  + [Hardware](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Hardware)
* [Safety](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Safety)
* Human&Practice
  + [collaboration](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Collaborations)
  + [Integrated Human&Practice](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Human_Practices)
* [Partnership](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Partnership)
* [Science Communication](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Education)
* Team
  + [member](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/member)
  + [Attributions](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/Attributions)
  + [contact](https://2020.igem.org/Team%3ABotchan_Lab_Tokyo/contact)

# Results

You can describe the results of your project and your future plans here.

### 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](http://parts.igem.org/Main_Page).** 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)