iGEM 2018 — Team Botchan_Lab_Tokyo

[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Botchan Lab Tokyo/Notebook

![](https://static.igem.org/mediawiki/2018/4/4f/2018_igem_mobile_menu_logo.svg)

![](https://static.igem.org/mediawiki/igem.org/3/3b/Menu_icon.svg)

[![](https://static.igem.org/mediawiki/2017/d/d8/T--Botchan_Lab_Tokyo--botchanlab_teamlogo.jpg)](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo)
[HOME](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo)

PROJECT

[RESULTS](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/Project)
[LABORATORY NOTEBOOK](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/Notebook)
[MODELING](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/Model)

LAB

[SAFETY](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/Safety)
[INTERLAB](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/InterLab)

PARTS

[COMPOSITE PARTS](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/Parts_Composite%20parts)
[PARTS BOOK](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/Parts_notebook)

HUMAN PRACTICE

[SILVER](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/HP/Silver)
[GOLD](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/Human_Practices)
[COLLABOLATIONS](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/Collaborations)

TEAM

[MEMBER](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/Team)
[ATTRIBUTION](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/Attributions)

## Lab notebook

---

#### 1.Protocol

We used 4 different strains

* *NifB*～*hesA*(9669 bp) *Paenibacillus polymyxa* ATCC 15970
* *NifVSU*(3324 bp) *Rahnella aquatilis* NBRC 105701
* *pheDH* (1189 bp) *Bacillus badius* NBRC 15713
* *gluDH*(1437 bp) *Bacillus licheniformis* NBRC 12200

![](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.

* pMW-f: cccggggatc ctctagagtc gacct

* pMW-r: gagctcgaat tcgtaatcat ggtca

* *nifVSU*-f(infusion): tgaccatgat tacgaattcg agctc GTACGACATC ACCCGAACCG

* *nifVSU*-r(infusion): aggtcgactc tagaggatcc ccggg TCAGCCTCCC TGAGCCTGAA

* *gluDH*-f:

  tcaggctcagggaggctgacccgggAGAAAGGGAGCTGAAAAAAGATG
* *gluDH*-r:

  tgcatgcctgcaggtcgactctagaTTAAACGACTCCCTGAGCGA

* *pheDH*-f :

  tcaggctcagggaggctgacccgggGGCTAAATAAAAGCGTTCAA

* *pheDH*-r:

  tgcatgcctgcaggtcgactctagaTTAGTTGCGAATATCCCAT

* *pMWnifVSU*-f:

  tctagagtcg acctgcaggc atgca

* *pMWnifVSU*-r:
  cccgggtcag cctccctgag cctga

※Capital letter is complementary to the ends of the linearized vector notebook.
We took over the last years project. So we want to check last year’s notebook before reading this year’s notebook.

 [Last year's notebookLB medium

|  |  |
| --- | --- |
| **Materials** | **Volume** |
| NaCl | 10 g |
| yeast extract | 5.0g |
| typtone | 10g |
| dH2O | 1L |
| pH 6.8～7.2 |  |

LB agar medium

|  |  |
| --- | --- |
| **Materials** | **Volume** |
| NaCl | 10 g |
| yeast extract | 5.0g |
| typtone | 10 g |
| Agarose | 1.5 % |
| dH2O | 1L |
| pH 6.8～7.2 |  |

Glutamic free medium　preparation
Glutamic free medium contain following

b>Glutamic free medium　preparation
Glutamic free medium contain following

```
Na2HPO4,      　　　      10.4g

KH2PO4,      　　　        3.4g

CaCl2·2H2O,      　　　    26mg

30 mg MgSO4,      　　　   30mg

MnSO4,      　　　        0.3mg

Ferric citrate,      　　　36mg

Na2MoO4·2H2O      　　　   7.6mg

p-aminobenzoic acid,      10μg

biotin      　　　         5μg

glucose                      4g

distilled water      　　　  1L
```

Nitrogen deficient medium　preparation
Nitrogen deficient medium contain following
Add Glutamate to Nitrogen Free medium by 5 mM.

### PCR or colony PCR

Mix PCR solutions and run the PCR machine in a program which is detailed below.

|  |  |  |  |  |  |  |  |  |
| --- | --- | --- | --- | --- | --- | --- | --- | --- |
| **PCR Solution** | Primer-F 10µM | template | Primer-R 10 µM | 2mM dNTPs | 2 x PCR Buffer | KOD Fx Neo DNA Polymerase | DW | Total |
| **Volume (µL)** | 1.5 | x | 1.5 | 10 | 25 | 1 | 11 | 50+x |

PCR
Cycle 1:(1 x) Step 1 94 ℃ for 2:00

Cycle 2:(35 x) Step１ 98 ℃ for 0:10 Step 2 68 ℃ for 08:00(*pMWnifVSU*)
for 2:00(*pheDH*,*gluDH*)

Cycle 3:(1 x) Step 1 4 ℃ for ∞

##### Electrophoresis

・Prepare agarose gel

1. Measure 0.2 g of agarose (1-2 %) to 20 mL of TAE in a beaker
2. Microwave until all agarose has dissolved
3. Allow agarose solution to cool down
4. Pour solution into gel tray.
5. Leave until gel has solidified then remove the com
6. Insert the gel into the electrophoresis chamber with the wells closest to the negative (black) electrode.
7. Gradually add 1xTBE (Tris-Borate-EDTA; electrophoresis buffer) to the chamber until the buffer just covers the top of the gel.
8. Add 2 µl of 6 x loading buffer to 5 µl of each sample.
9. Add 5 µl of each sample with 2 µl of 10 x loading buffer and 0.5~5 kb marker .
10. Start electrophoresis at 100 V.
11. Stop at appropriate time.
12. Put the gel in the container which is filled with EtBr

##### Cloning

We used in-fusion cloning

|  |  |
| --- | --- |
| 5X In-Fusion HD Enzyme Premix | ２µl |
| Linearized vector | 4 µl |
| PCR fragment | 4 µl |
| total volume 10 µl | |

+ We refined PCR mixture by GFX　pulification kit.
+ Set up the in-Fusion cloning reaction.](https://2017.igem.org/Team%3ABotchan_Lab_Tokyo/Notebook)

* Incubate the reaction for 15 min at 50 °C, then place on ice.
* store the cloning reaction at refrigerator(-20 ℃) over night.

##### Transformation

1. Thaw competent cells(JM109) on ice
2. Add plasmid DNA (1 µl) to competent cells. leave them on ice for 30 minutes
3. Keep them in heating block(42 ℃) for 45 seconds, then cool on ice for 1 minutes
4. Add 500 µl SOC liquid culture medium, then incubate with shaking for 1hour at 37 °C
5. Seed cells onto LB+ampicillin agar plate. Incubate for 24 hours at 37 ℃

##### Gel purification

We used illustra GFX PCR DNA and Gel Band Purification Kit

１.Sample Capture

* Using a clean scalpel, long wavelength (365 nm) ultraviolet light and minimal exposure time, cut out an agarose band containing the sample of interest.
* Add 10 µl Capture buffer type 3 for each 10 mg of gel slice, for example, add 300 µl Capture buffer type 3 to each 300 mg gel slice.
* Mix by inversion and incubate at 60 °C for 15–30 minutes until the agarose is completely dissolved. Mix by inversion every 3 minutes.
* For each purification that is to be performed, place one GFX MicroSpin column into one Collection tube.

２. Sample Binding

* Centrifuge Capture buffer type 3- sample mix briefly to collect the liquid at the bottom of the tube.
* Transfer up to 800 µl Capture buffer type 3- sample mix onto the assembled GFX MicroSpin column and Collection tube.
* Incubate at room temperature for 1 minute.
* Spin the assembled column and Collection tube at 16,000 × g for 30 seconds.
* Discard the flow through by emptying the Collection tube. Place the GFX MicroSpin column back inside the Collection tube.
* f. Repeat Sample Binding steps b. to e. as necessary until all sample is loaded.

３. Wash & Dry

* a. Add 500 µl Wash buffer type 1 to the GFX MicroSpin column.
* b. Spin the assembled column and Collection tube at 16,000 × g for 30 seconds
* c. Discard the Collection tube and transfer the GFX MicroSpin column to a fresh DNase-free 1.5 ml microcentrifuge tube (supplied by user).

４.Elution

1. a. Add 10–50 µl Elution buffer type 4 or type 6 to the center of the membrane in the assembled GFX MicroSpin column and sample Collection tube.
2. b. Incubate the assembled GFX MicroSpin column and sample Collection tube at room temperature for 1 minute.
3. c. Spin the assembled column and sample Collection tube at 16,000 × g for 1 minute to recover the purified DNA.
4. d. Proceed to downstream application. Store the purified DNA at -20 °C.

##### Purification of plasmid DNA

We used QIAprep Spin Miniprep Kit

1. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
2. Add 250 μl Buffer P2 and gently invert the tube 4–6 times to mix.
3. Add 350 μl Buffer N3 and invert the tube immediately but gently 4–6 times.
4. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
5. Apply the supernatants from step 4 to the QIAprep spin column by decanting or pipetting.
6. Centrifuge for 30–60 s. Discard the flow-through.
7. (Optional): Wash the QIAprep spin column by adding 0.5 ml Buffer PB and centrifuging for 30–60 s. Discard the flow-through.
8. Wash QIAprep spin column by adding 0.75 ml Buffer PE and centrifuging for 30–60 s.
9. Discard the flow-through, and centrifuge for an additional 1 min to remove residual wash buffer.
10. Place the QIAprep column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM Tris・Cl, pH 8.5) to the center of each QIAprep spin column, let stand for 1 min, and centrifuge for 1 min.

※pre-heat Buffer EB (or water) to 70 °C prior to eluting DNA

##### Restriction digests

We used sac1 and Xma1 as restriction enzyme.

1. Add 68 ul of DNA to be digested into a 1.5 ml microcentrifuge tube.
2. Add 10 ul of 10x buffer.
3. Add 20 ul of pSMW218 or PCR product.
4. Add 2.0 ul of Sac1.
5. Incubate the restriction digest at 37 ℃ for 5 hour.

##### Purification of DNA from an enzymatic reaction by GFX　pulification kit.

1. Add 68 ul of DNA to be digested into a 1.5 ml microcentrifuge tube.
2. Add 10 ul of 10x buffer.
3. Add 20 ul of pSMW218 or PCR product.
4. Add 2.0 ul of Xma1.
5. Incubate the restriction digest at 37 ℃ for overnight.

Purification of DNA from an enzymatic reaction by GFX pulification kit.

##### Purification of DNA from PCR mixture

* a.Add 500 μL of capture buffer type 3 to 100 μL of sample.
* b.Pipetting
* c.Centrifuge for 1 min at 15,000 rpm (~17,900 x g).
* d.Discard the flow through
* e.Add 500 μL　of wash buffer.
* f. Centrifuge for 1 min at 15,000 rpm (~17,900 x g)

##### Indophenol

Day1

* a. The samples are planted in 5 ml of antibiotic LB medium.
* b.Incubate 24 h at 30 ℃

Day2

* c. 180 µL of incubated LB medium and samples are added to nitrogen free medium.
* d. Add Glutamate as nitrogen source(Nitrogen deficient medium)
* e .Add appropriate antibiotics.
* f. If sample has pMW plasmid, Add IPTG to activate Lac promoter. Add MSX if necessary.
* e. Fill Nitrogen into Nitrogen deficient medium.
* g.Incubate at 30 ℃

Day3~5

* h.Add indophenol reagent.
* I.Wait 30 minitues.
* J .Measure abs 600

##### Indophenol reagent

| Materials | Volume (µl) |
| --- | --- |
| Phenol/EtOH | 40 |
| Sodium nitroprusside/H2O | 40 |
| Sodium hypochlorite | 100 |

#### 2.Laboratory notebook 5/21LB medium preparation LB agar medium preparation(Anitibiotics:Amp,Km,Amp+Km) 5/22LB agar medium preparation LB agar medium preparation 5/24 Transformation | Sample Name | Sample Volume (μl) | Competent cell(E.coli)/(μl) | Medium | | --- | --- | --- | --- | | pHY*nifB,H,D,K,N,X,hesA* | 1 | 20 | SOC Medium | | pMW*nifVSU* | 1 | 20 | SOC Medium | | pHY*nifB,H,D,K,N,X,hesA+pHYnifVSU* | 1 | 20 | SOC Medium | | pHY | 1 | 20 | SOC Medium | | pHYpMW | 0.5+0.5 | 20 | SOC Medium | 5/25Preculture Transformant is planted in 5 ml of LB medium and streaked to antibiotic LB agar medium ~Failed~ 5/30 Preculture | Name | medium | Antibiotics | | --- | --- | --- | | *E.coli*(pHY) | LB | Amp | | *E.coli*(pHY*nifB,H,D,K,N,X,hesA*) | LB | Amp | | *E.coli*(pMW) | LB | Km | | *E.coli*(pMW*nifB,H,D,K,N,X,hesA*) | LB | Km | | *E.coli*(pHYpMW) | LB | Amp,Km | | *E.coli*(pHY*nifB,H,D,K,N,X,hesA*,pMW*nifVSU*) | LB | Amp,Km | 5/31Plasmid Extraction Sample: Precultured sample 6/1 Electrophresis | Lane | sample/(µl) | | --- | --- | | 1.pHY | 5 | | 2.pHY*nif* | 5 | | 3.pMW | 5 | | 4.pMW*nif* | 5 | | 5.pHYpMW | 5 | | 6.pHY*nifB,H,D,K,N,X,hesA*,pMW*nifVSU* | | ![](https://static.igem.org/mediawiki/2018/7/73/T--Botchan_Lab_Tokyo--D0601.png) 6/4 Electrophresis | Lane | sample/(µl) | | --- | --- | | 1.pMW | 5 | | 2.pMW | 5 | | 3.pMW*nifVSU* | 5 | | 4.pMW*nifVSU* | 5 | | 5.pHY*nifB,H,D,K,N,X,hesA*,pMW*nifVSU* | 5 | ![](https://static.igem.org/mediawiki/2018/d/dc/T--Botchan_Lab_Tokyo--D0604.png) 6/4LB medium preparation 6/6 Transformation | Sample Name | Sample Volume (µl) | Competent Cells(JM109)/(µl) | Medium | | --- | --- | --- | --- | | pHY | 1 | 20 | SOC Medium | | pHY | 1 | 20 | SOC Medium | 6/11LB agar medium preparation(Antibiotics: Amp) 6/14 Transformation | Sample Name | Sample Volume (µl) | Competent Cells(JM109)/(µl) | Medium | | --- | --- | --- | --- | | pHY | 1 | 20 | SOC Medium | | pHY | 1 | 20 | SOC Medium | 6/18LB agar prepararation 6/25 Colony PCR | Templates/(µl) | Primers/(µl) | Primer's name | MilliQ/(µl) | PreMix/(µl) | Total/(µl) | | --- | --- | --- | --- | --- | --- | | pMW | 1.5 | pMW | 1 | 50 | 51 | | pMW | 1.5 | pMW | 1 | 50 | 51 | | *Rahenella aquatilis* | 1.5 | *nifVSU* | 1 | 50 | 51 | | *Rahenella aquatilis* | 1.5 | *nifVSU* | 1 | 50 | 51 | | *Rahenella aquatilis* | 1.5 | *nifVSU* | 1 | 50 | 51 | | *Rahenella aquatilis* | 1.5 | *nifVSU* | 1 | 50 | 51 | 6/26 Electrophresis | Lane | sample/µl) | | --- | --- | | 1..pMW | 5 | | 2..pMW | 5 | | 3.*nifVSU* for restriction enzyme | 5 | | 4.*nifVSU* for restriction enzyme | 5 | | 5.*nifVSU* for infusion | 5 | | 6.*nifVSU* for infusion | 5 | ![](https://static.igem.org/mediawiki/2018/4/4c/T--Botchan_Lab_Tokyo--D0626.png) 6/28 Electrophresis | Lane | sample/(µl) | | --- | --- | | 1.pMW | 5 | | 2.pMW | 5 | | 3.*nifVSU* for restriction enzyme | 5 | | 4.*nifVSU* for restriction enzyme | 5 | ![](https://static.igem.org/mediawiki/2018/0/0b/T--Botchan_Lab_Tokyo--D0627.png) 7/2Purification of DNA from PCR mixture Sample: June26,sample5 June 28,sample2 June 28,sample4 7/3Infusion Cloning pMW and *nifVSU* 7/3 Electrophresis | Lane | sample/(µl) | | --- | --- | | 1.*nifVSU* | 5 | | 2.pMW | 5 | | 3.pHY | 5 | | 4.none | 5 | | 5.*nifVSU* | 5 | | 6.pMW | 5 | ![](https://static.igem.org/mediawiki/2018/a/a3/T--Botchan_Lab_Tokyo--D0703.png) 7/4 Transformation | Sample Name | Sample Volume (µl) | Competent Cells(JM109)/(µl) | Medium | | --- | --- | --- | --- | | pMW | 1 | 20 | SOC Medium | | pMW*nifVSU* | 1 | 20 | SOC Medium | 7/5pMW streaked to antibiotic LB agar medium 7/6Km,amp, stock tube preparation 7/9Anitibiotick check 7/10 Transformation | Sample Name | Sample Volume (µl) | Competent Cells(JM109)/(µl) | Medium | | --- | --- | --- | --- | | pMW | 1 | 20 | SOC Medium | | pMW*nifVSU* | 1 | 20 | SOC Medium | 7/11 Colony PCR | Templates/(µl) | Primers/(µl) | Primer's name | MilliQ/(µl) | PreMix/(µl) | Total/(µl) | | --- | --- | --- | --- | --- | --- | | *Rahnella aquatillis* | 1.5 | *nifVSU* | 1 | 50 | 51 | | pMW | 1.5 | *nifVSU* | 1 | 50 | 51 | | pMW*nifVSU* | 1.5 | *nifVSU* | 1 | 50 | 51 | | pMW*nifVSU* | 1.5 | *nifVSU* | 1 | 50 | 51 | | pMW*nifVSU* | 1.5 | *nifVSU* | 1 | 50 | 51 | | pMW*nifVSU* | 1.5 | *nifVSU* | 1 | 50 | 51 | 7/12 Electrophresis | Lane | sample/(µl) | | --- | --- | | 1.*NifV,S,U* | 5 | | 2.*NifV,S,U* | 5 | | 3.*NifV,S,U* | 5 | | 4.*NifV,S,U* | 5 | | 5.*NifV,S,U* | 5 | | 6.*NifV,S,U* | 5 | ![](https://static.igem.org/mediawiki/2018/6/61/T--Botchan_Lab_Tokyo--D0712.png) 7/12Preculture for plasmid extraction Sample: 1.JM109(pMW) 2.Transformant(pMW*nifVSU*) 1 7/13 Electrophresis | Lane | sample/(µl) | | --- | --- | | 1.*pMW* | 5 | | 2.*pMWVSU* | 5 | | 3.*pMW Sac1* | 5 | | 4.*VSU Sac1 pMW* | 5 | | 5.*pMW Xma1* | 5 | | 6.*VSU Xma1 pMW* | 5 | ![](https://static.igem.org/mediawiki/2018/9/9b/T--Botchan_Lab_Tokyo--D0713.png) 8/9 Transformation | Sample Name | Sample Volume (µl) | Competent Cells(JM109)/(µl) | Medium | | --- | --- | --- | --- | | pMW*pHY* | 0.5+0.5 | 20 | SOC Medium | | pMW*nifVSU* +pHY*nifB,H,D,K,N,X,hesA* | 0.5+0.5 | 20 | SOC Medium | 8/21 Transformation | Sample Name | Sample Volume (µl) | Competent Cells(JM109)/(µl) | Medium | | --- | --- | --- | --- | | pHY | 0.5+0.5 | 20 | SOC Medium | | pHY*nifB,H,D,K,N,X,hesA*,pMW | 0.5+0.5 | 20 | SOC Medium | | pHY+pMW | 0.5+0.5 | 20 | | 8/22Preculture for plasmid extractionElectrophoresis | Lane | sample/(µl) | | --- | --- | | 1.pHY*nifB~hesA*pMW*nifVSU* | 5 | | 2.pHY,pMW | 5 | | 3.pHY | 5 | | 4.pHY*nifB~hesA* | 5 | | 5.pMW | 5 | | 6.pMW*nifVSU* | 5 | ![](https://static.igem.org/mediawiki/2018/a/ab/T--Botchan_Lab_Tokyo--D0822.png) 8/30 Colony PCR | Templates/(µl) | Primers/(µl) | Primer's name | MilliQ/(µl) | PreMix/(µl) | Total/(µl) | | --- | --- | --- | --- | --- | --- | | pMW*nifVSU* | 1.5 | pMW*nifVSU* | 1 | 50 | 51 | | pMW*nifVSU* | 1.5 | pMW*nifVSU* | 1 | 50 | | 9/6 Colony PCR | Templates/(µl) | Primers/(µl) | Primer's name | MilliQ/(µl) | PreMix/(µl) | Total/(µl) | | --- | --- | --- | --- | --- | --- | | *Bacilllus lichenformis* | 1.5 | *gluDH* | 1 | 50 | 51 | | *Bacilllus lichenformis* | 1.5 | *gluDH* | 1 | 50 | 51 | | *Bacilllus lichenformis* | 1.5 | *gluDH* | 1 | 50 | 51 | | *Bacilllus lichenformis* | 1.5 | *gluDH* | 1 | 50 | 51 | | pMW*VSU* extracted from transformant made July 10 | 1.5 | *gluDH* | 1 | 50 | 51 | | pMW*VSU* extracted from transformant made July 10 | 1.5 | *gluDH* | 1 | 50 | | 9/10Gel extraction Sample: September 6 Sample2 9/11Electrophoresis sample:purified *nifVSU* ![](https://static.igem.org/mediawiki/2018/b/b3/T--Botchan_Lab_Tokyo--D0911.png) Transformation | Sample Name | Sample Volume (µl) | Competent Cells(JM109)/(µl) | Medium | | --- | --- | --- | --- | | PMW*nifVSU+pheDH* | 0.5+0.5 | 20 | SOC Medium | | PMW*nifVSU+gluDH* | 0.5+0.5 | 20 | SOC Medium | | pMW*nifVSU+gluDH* | 0.5+0.5 | 20 | SOC Medium | 9/12Preculture of Transformant9/13Plasmid extraction 1.pMW*pheDH* 2.pMW*pheDH*+pHY*nif* 3.pMW 4.pMW+pHY 9/14Restriction enzyme digest 9/17 | Lane | sample/(µl) | | --- | --- | | 1.*gluDH* | 5 | | 2.*gluDH* | 5 | | 3.pMW*nifVSU* | 5 | | 4.pMW*nifVSU* | 5 | ![](https://static.igem.org/mediawiki/2018/6/60/T--Botchan_Lab_Tokyo--D0917.png) 9/20 Colony PCR | Templates/(µl) | Primers/(µl) | Primer's name | MilliQ/µl) | PreMix/(µl) | Total/(µl) | | --- | --- | --- | --- | --- | --- | | *Bacilllus lichenformis* | 1.5 | *gluDH* | 1 | 50 | 51 | | *Bacilllus lichenformis* | 1.5 | *gluDH/i>* | 1 | 50 | 51 | | JM109(pMW*nifVSU*) | 1.5 | pMW*nifVSU* | 1 | 50 | 51 | | JM109(pMW*nifVSU*) | 1.5 | pMW*nifVSU* | 1 | 50 | 51 | 9/21 Electrophresis | Lane | sample/(µl) | | --- | --- | | 1.*gluDH* | 5 | | 2.*gluDH* | 5 | | 3.pMW*nifVSU* | 5 | | 4.none | 5 | | 5.pMW*nifVSU* | 5 | | 6.none | 5 | ![](https://static.igem.org/mediawiki/2018/d/dc/T--Botchan_Lab_Tokyo--D0920.png) 9/25DNA extraction from Agarose gel Sample: 1.pMW*nifVSU* 2.pMW*nifVSU* Infusion cloning Sample:pMW*nifVSU*+*gluDH* 10/8 transformation | Sample Name | Sample Volume (µl) | Competent Cells/(µl) | Medium | | --- | --- | --- | --- | | pMW*nifVSUgluDH* | 1.0 | 20 | SOC | | pHY*nifB*~*hesA*+pMW*nifVSUgluDH* | 0.5,0.5 | 20 | SOC | | pMW(positive control) | 1.0 | 20 | SOC | 10/10 Preculture for plasmid extraction | Name | medium | Antibiotics | | --- | --- | --- | | E.coli(pMW*nifVSUgluDH*) | LB | Km | | E.coli(pHY*nifB*~*hesA*,pMW*nifVSUgluDH*) | LB | Amp,Km | Indophenol method 5/23Nitrogen free medium A,B,C and D preparation 6/19 Preculture for indophenol | Name | medium | Antibiotics | | --- | --- | --- | | *Paenibacillus polymyxa* | LB | none | | *E.coli* | LB | none | 6/20 Inoculation | Sample Name | glutamate(mM) | Antibiotics | 100mM IPTG(µl) | 50mM MSX(µl) | Nitrgen free medium(ml) | | --- | --- | --- | --- | --- | --- | | *Paenibacillus polymyxa* | 25 | none | 0 | 0 | 2.5 | | *E.coli* | 25 | none | ２５0 | 0 | 2.5 | | *Paenibacillus polymyxa* | 25 | none | 0 | 0 | 2.5 | | *E.coli* | 25 | none | 0 | 0 | 2.5 | 6/20 Inoculation | Sample Name | Phenol/EtOH(µl) | Sodium nitroprusside/H2O(µl) | Sodium hypochlorite(µl) | | --- | --- | --- | --- | | *Paenibacillus polymyxa* | 40 | 40 | 100 | | *E.coli* | 40 | 40 | 100 | | *Paenibacillus polymyxa* | 40 | 40 | 100 | | *E.coli* | 40 | 40 | 100 | 6/21 Indophenol(24h) Kasahara | Sample Name | Phenol/EtOH(µl) | Sodium nitroprusside/H2O(µl) | Sodium hypochlorite(µl) | | --- | --- | --- | --- | | *Paenibacillus polymyxa* | 40 | 40 | 100 | | *E.coli* | 40 | 40 | 100 | | *Paenibacillus polymyxa* | 40 | 40 | 100 | | i>E.coli | 40 | 40 | | 6/22 Indophenol(48h) Kasahara | Sample Name | Phenol/EtOH(µl) | Sodium nitroprusside/H2O(µl) | Sodium hypochlorite(µl) | | --- | --- | --- | --- | | *Paenibacillus polymyxa* | 40 | 40 | 100 | | *E.coli* | 40 | 40 | 100 | | *Paenibacillus polymyxa* | 40 | 40 | 100 | | *E.coli* | 40 | 40 | | 9/2 Preculture for indophenol | Sample Name | medium | Antibiotics | | --- | --- | --- | | *E.coli*(pHY*nifB*~*hesA*) | LB | Amp | | *E.coli*(pHY*nifB*~*hesA*+pMW*nifVSU*) | LB | Amp+Km | | *E.coli*(pHY) | LB | Amp | | *E.coli*(pHY*nifB*~*hesA*+pMW) | LB | Amp+Km | | *E.coli*(pHY+pMW) | LB | Amp+Km | | *Paenibacillus polymyxa* | LB | none | 9/3 Inoculation | Sample Name | glutamate(mM) | Antibiotics | 100mM IPTG(µl) | 50mM MSX(µl) | Nitrgen free medium(ml) | | --- | --- | --- | --- | --- | --- | | *E.coli*(pHY*nifB*~*hesA*)48 h | 25 | Amp | 0 | 0 | 2.5 | | *E.coli*(pHY*nifB*~*hesA*)72 h | 25 | Amp | 0 | 0 | 2.5 | | *E.coli*(pHY*nifB*~*hesA*+pMW*nifVSU*)48h | 25 | Amp,Km | 25 | 0 | 2.5 | | *E.coli*(pHY*nifB*~*hesA*+pMW*nifVSU*)72 h | 25 | Amp,Km | 25 | 0 | 2.5 | | *E.coli*(pHY)48 h | 25 | Amp | 0 | 0 | 2.5 | | *E.coli*(pHY)72 h | 25 | Amp | 0 | 0 | 2.5 | | *E.coli*(pHY*nifB*~*hesA*+pMW)48 h | 25 | Amp,Km | 25 | 0 | 2.5 | | *E.coli*(pHY*nifB*~*hesA*+pMW)72 h | 25 | Amp,Km | 25 | 0 | 2.5 | | *E.coli*(pHY+pMW)48 h | 25 | Amp,Km | 25 | 0 | 2.5 | | *E.coli*(pHY+pMW)72 h | 25 | Amp,Km | 25 | 0 | 2.5 | | *Paenibacillus polymyxa* 48 h | 25 | none | 0 | 0 | 2.5 | | *Paenibacillus polymyxa* 72 h | 25 | none | 0 | 0 | 2.5 | | Medium only | 25 | none | 0 | 0 | 2.5 | | Medium only | 25 | none | 0 | 0 | 2.5 | | *E.coli*(pHY*nifB*~*hesA*,pMW*nifVSU*)48 h MSX | 25 | Amp,Km | 25 | 25 | 2.5 | | *E.coli*(pHY*nifB*~*hesA*,pMW*nifVSU*)72 h MSX | 25 | Amp,Km | 25 | 25 | 2.5 | | *E.coli*(pHY+pMW)48 h MSX | 25 | Amp,Km | 25 | 25 | 2.5 | | *E.coli*(pHY+pMW)72 h MSX | 25 | Amp,Km | 25 | 25 | 2.5 | | *Paenibacillus polymyxa* 48 h MSX | 25 | none | 0 | 25 | 2.5 | | *Paenibacillus polymyxa* 72 h MSX | 25 | none | 0 | | 2.5 | 9/5 Indophenol(48 h) | Sample Name | Phenol/EtOH(µl) | Sodium nitroprusside/H2O(µl) | Sodium hypochlorite(µl) | | --- | --- | --- | --- | | 1.*E.coli*(pHY*nifB*~*hesA*) | 40 | 40 | 100 | | 2.*E.coli*(pHY*nifB*~*hesA*+pMW*nifVSU*) | 40 | 40 | 100 | | 3.*E.coli*(pHY) | 40 | 40 | 100 | | 4.*E.coli*(pHY*nifB*~*hesA*+pMW) | 40 | 40 | 100 | | 5.*E.coli*(pHY+pMW) | 40 | 40 | 100 | | 6.*Paenibacillus polymyxa* | 40 | 40 | 100 | | 7.Medium only | 40 | 40 | 100 | ![](https://static.igem.org/mediawiki/2018/2/2d/T--Botchan_Lab_Tokyo--K090548.png) Indophenol(48 h)MSX | Sample Name | Phenol/EtOH(µl) | Sodium nitroprusside/H2O(µl) | Sodium hypochlorite(µl) | | --- | --- | --- | --- | | 1.*E.coli*(pHY*nifB*~*hesA*+pMW*nifVSU*) | 40 | 40 | 100 | | 2.*E.coli*(pHY+pMW) | 40 | 40 | 100 | | 3.*Paenibacillus polymyxa* | 40 | 40 | 100 | | 4.Medium only | 40 | 40 | 100 | ![](https://static.igem.org/mediawiki/2018/archive/9/9d/20181016060043%21T--Botchan_Lab_Tokyo--K090548M.png) 9/6 Indophenol(72 h) | Sample Name | Phenol/EtOH(µl) | Sodium nitroprusside/H2O(µl) | Sodium hypochlorite(µl) | | --- | --- | --- | --- | | 1.*E.coli*(pHY*nifB*~*hesA*) | 40 | 40 | 100 | | 2.*E.coli*(pHY*nifB*~*hesA*+pMW*nifVSU*) | 40 | 40 | 100 | | 3.*E.coli*(pHY) | 40 | 40 | 100 | | 4.*E.coli*(pHY*nifB*~*hesA*+pMW) | 40 | 40 | 100 | | 5.*E.coli*(pHY+pMW) | 40 | 40 | 100 | | 6.*Paenibacillus polymyxa* | 40 | 40 | 100 | | 7.Medium only | 40 | 40 | 100 | ![](https://static.igem.org/mediawiki/2018/2/21/T--Botchan_Lab_Tokyo--K090672.png) Indophenol(72 h)MSX | Sample Name | Phenol/EtOH(µl) | Sodium nitroprusside/H2O(µl) | Sodium hypochlorite(µl) | | --- | --- | --- | --- | | 1.*E.coli*(pHY*nifB*~*hesA*+pMW*nifVSU*) | 40 | 40 | 100 | | 2.*E.coli*(pHY+pMW) | 40 | 40 | 100 | | 3.*Paenibacillus polymyxa* | 40 | 40 | 100 | | 4.Medium only | 40 | 40 | 100 | ![](https://static.igem.org/mediawiki/2018/6/68/T--Botchan_Lab_Tokyo--K090672M.png) 9/10 Preculture for indophenol | Sample Name | Medium | Antibiotics | | --- | --- | --- | | *E.coli*(pHY*nifB*~*hesA*) | LB | Amp | | *E.coli*(pHY*nifB*~*hesA*+pMW*nifVSU*) | LB | Amp,Km | | *E.coli*(pHY) | LB | Amp | | *E.coli*(pHY*nifB*~*hesA*+pMW) | LB | Amp,Km | | *E.coli*(pHY+pMW) | LB | Aap,Km | | *Paenibacillus polymyxa* | LB | none | | Medium only | LB | none | 9/11 Inoculation | Sample Name | glutamate(mM) | Antibiotics | 100mM IPTG(µl) | 50mM MSX(µl) | Nitrgen free medium(ml) | | --- | --- | --- | --- | --- | --- | | *E.coli*(pHY*nifB*~*hesA*) | 25 | Amp | 0 | 0 | 2.5 | | *E.coli*(pHY*nifB*~*hesA*+pMW*nifVSU*) | 25 | Amp,Km | 25 | 0 | 2.5 | | *E.coli*(pHY) | 25 | Amp | 0 | 0 | 2.5 | | *E.coli*(pHY*nifB*~*hesA*+pMW) | 25 | Amp,Km | 25 | 0 | 2.5 | | *E.coli*(pHY+pMW) | 25 | Amp,Km | 25 | 0 | 2.5 | | *Paenibacillus polymyxa* | 25 | none | 0 | 0 | 2.5 | | medium only | 25 | none | 0 | 0 | 2.5 | | *E.coli*(pHY*nifB*~*hesA*+pMW*nifVSU*) MSX | 25 | Amp,Km | 25 | 25 | 2.5 | | *E.coli*(pHY) MSX | 25 | Amp | 0 | 25 | 2.5 | | *E.coli*(pHY*nifB*~*hesA*+pMW) MSX | 25 | Amp,Km | 25 | 25 | 2.5 | | *E.coli*(pHY+pMW) MSX | 25 | Amp,Km | 25 | 25 | 2.5 | | MSX | 25 | none | 0 | 25 | 2.5 | 9/14 Indopheol(72h) | Sample Name | Phenol/EtOH(µl) | Sodium nitroprusside/H2O(µl) | Sodium hypochlorite(µl) | | --- | --- | --- | --- | | 1.*E.coli*(pHY*nifB*~*hesA*) | 40 | 40 | 100 | | 2.*E.coli*(pHY*nifB*~*hesA*+pMW*nifVSU*) | 40 | 40 | 100 | | 3.*E.coli*(pHY) | 40 | 40 | 100 | | 4.*E.coli*(pHY*nifB*~*hesA*+pMW) | 40 | 40 | 100 | | 5.*E.coli*(pHY+pMW) | 40 | 40 | 100 | | 6.*Paenibacillus polymyxa* | 40 | 40 | 100 | | 7.medium only | 40 | 40 | 100 | ![](https://static.igem.org/mediawiki/2018/5/5e/T--Botchan_Lab_Tokyo--K091472.png) Indopheol(72h)MSX | Sample Name | Phenol/EtOH(µl) | Sodium nitroprusside/H2O(µl) | Sodium hypochlorite(µl) | | --- | --- | --- | --- | | 1.*E.coli*(pHY*nifB*~*hesA*+pMW*nifVSU*) MSX | 40 | 40 | 100 | | 2.*E.coli*(pHY) MSX | 40 | 40 | 100 | | 3.*E.coli*(pHY*nifB*~*hesA*+pMW) MSX | 40 | 40 | 100 | | 4.*E.coli*(pHY+pMW) MSX | 40 | 40 | 100 | | 5.*Paenibacillus polymyxa* MSX | 40 | 40 | 100 | ![](https://static.igem.org/mediawiki/2018/1/17/T--Botchan_Lab_Tokyo--K091472M.png) 9/24 Preculture for indophenol | Sample Name | medium | Antibiotics | | --- | --- | --- | | *E.coli*(pHY*nifB*~*hesA*) | LB | Amp | | *E.coli*(pHY*nifB*~*hesA*+pMW*nifVSU*) | LB | Amp,Km | | *E.coli*(pHY) | LB | Amp | | *E.coli*(pHY*nifB*~*hesA*+pMW) | LB | Amp,Km | | *E.coli*(pHY+pMW) | LB | Aap,Km | | *Paenibacillus polymyxa* | LB | none | | Medium only | LB | none | 9/25 Inoculation | Sample Name | glutamate(mM) | Antibiotics | 100 mM IPTG(&microl) | 50 mM MSX(&microl) | Nitrgen free medium(ml) | | --- | --- | --- | --- | --- | --- | | *E.coli*(pHY*nifB*~*hesA*)48 h | 25 | Amp | 0 | 0 | 2.5 | | *E.coli*(pHY*nifB*~*hesA*+pMW*nifVSU*)48h | 25 | Amp,Km | 25 | 0 | 2.5 | | *E.coli*(pHY)48 h | 25 | Amp | 0 | 0 | 2.5 | | *E.coli*(pHY*nifB*~*hesA*+pMW)48 h | 25 | Amp,Km | 25 | 0 | 2.5 | | *E.coli*(pHY+pMW)48 h | 25 | Amp,Km | 25 | 0 | 2.5 | | *Paenibacillus polymyxa* 48 h | 25 | none | 0 | 0 | 2.5 | | Medium only | 25 | none | 0 | 0 | 2.5 | | *E.coli*(pHY*nifB*~*hesA*,pMW*nifVSU*)48 h MSX | 25 | Amp,Km | 25 | 25 | 2.5 | | *E.coli*(pHY+pMW)48 h MSX | 25 | Amp,Km | 25 | 25 | 2.5 | 9/28 Indophenol(48h)-1 | Sample Name | Phenol/EtOH(&microl) | Sodium nitroprusside/H2O(&microl) | Sodium hypochlorite(&microl) | | --- | --- | --- | --- | | 1.*E.coli*(pHY*nifB*~*hesA*) | 40 | 40 | 100 | | 2.*E.coli*(pHY*nifB*~*hesA*+pMW*nifVSU*) | 40 | 40 | 100 | | 3.*E.coli*(pHY) | 40 | 40 | 100 | | 4.*E.coli*(pHY*nifB*~*hesA*+pMW) | 40 | 40 | 100 | | 5.*E.coli*(pHY+pMW) | 40 | 40 | 100 | | 6.*Paenibacillus polymyxa* | 40 | 40 | 100 | | 7.Medium only | 40 | 40 | | ![](https://static.igem.org/mediawiki/2018/8/8a/T--Botchan_Lab_Tokyo--K092848_1.png) Indophenol(48h)MSX-1 | Sample Name | Phenol/EtOH(&microl) | Sodium nitroprusside/H2O(&microl) | Sodium hypochlorite(&microl) | | --- | --- | --- | --- | | 1.*E.coli*(pHY*nifB*~*hesA*+pMW*nifVSU*) MSX | 40 | 40 | 100 | | 2.*E.coli*(pHY+pMW)MSX | 40 | 40 | 100 | ![](https://static.igem.org/mediawiki/2018/f/fd/T--Botchan_Lab_Tokyo--K0928M_1.png) 9/28 Indophenol(48h)-2 | Sample Name | Phenol/EtOH(&microl) | Sodium nitroprusside/H2O(&microl) | Sodium hypochlorite(&microl) | | --- | --- | --- | --- | | 1.*E.coli*(pHY*nifB*~*hesA*) | 40 | 40 | 100 | | 2.*E.coli*(pHY*nifB*~*hesA*+pMW*nifVSU*) | 40 | 40 | 100 | | 3.*E.coli*(pHY) | 40 | 40 | 100 | | 4.*E.coli*(pHY*nifB*~*hesA*+pMW) | 40 | 40 | 100 | | 5.*E.coli*(pHY+pMW) | 40 | 40 | 100 | | 6.*Paenibacillus polymyxa* | 40 | 40 | 100 | | 7.Medium only | 40 | 40 | | ![](https://static.igem.org/mediawiki/2018/1/1b/T--Botchan_Lab_Tokyo--K092848_2.png) Indophenol(48h)MSX-2 | Sample Name | Phenol/EtOH(&microl) | Sodium nitroprusside/H2O(&microl) | Sodium hypochlorite(&microl) | | --- | --- | --- | --- | | 1.*E.coli*(pHY*nifB*~*hesA*+pMW*nifVSU*) MSX | 40 | 40 | 100 | | 2.*E.coli*(pHY+pMW)MSX | 40 | 40 | 100 | ![](https://static.igem.org/mediawiki/2018/8/8a/T--Botchan_Lab_Tokyo--K092872M2.png) 9/28 Indophenol(48h)-3 | Sample Name | Phenol/EtOH(&microl) | Sodium nitroprusside/H2O(&microl) | Sodium hypochlorite(&microl) | | --- | --- | --- | --- | | 1.*E.coli*(pHY*nifB*~*hesA*) | 40 | 40 | 100 | | 2.*E.coli*(pHY*nifB*~*hesA*+pMW*nifVSU*) | 40 | 40 | 100 | | 3.*E.coli*(pHY) | 40 | 40 | 100 | | 4.*E.coli*(pHY*nifB*~*hesA*+pMW) | 40 | 40 | 100 | | 5.*E.coli*(pHY+pMW) | 40 | 40 | 100 | | 6.*Paenibacillus polymyxa* | 40 | 40 | 100 | | 7.Medium only | 40 | 40 | | ![](https://static.igem.org/mediawiki/2018/5/58/T--Botchan_Lab_Tokyo--K092848_3.png) Indophenol(48h)MSX-3 | Sample Name | Phenol/EtOH(&microl) | Sodium nitroprusside/H2O(&microl) | Sodium hypochlorite(&microl) | | --- | --- | --- | --- | | 1.*E.coli*(pHY*nifB*~*hesA*+pMW*nifVSU*) MSX | 40 | 40 | 100 | | 2.*E.coli*(pHY+pMW)MSX | 40 | 40 | 100 | ![](https://static.igem.org/mediawiki/2018/4/47/T--Botchan_Lab_Tokyo--K092848_4.png) 9/28 Indophenol(48h)-4 | Sample Name | Phenol/EtOH(&microl) | Sodium nitroprusside/H2O(&microl) | Sodium hypochlorite(&microl) | | --- | --- | --- | --- | | 1.*E.coli*(pHY*nifB*~*hesA*) | 40 | 40 | 100 | | 2.*E.coli*(pHY*nifB*~*hesA*+pMW*nifVSU*) | 40 | 40 | 100 | | 3.*E.coli*(pHY) | 40 | 40 | 100 | | 4.*E.coli*(pHY*nifB*~*hesA*+pMW) | 40 | 40 | 100 | | 5.*E.coli*(pHY+pMW) | 40 | 40 | 100 | | 6.*Paenibacillus polymyxa* | 40 | 40 | 100 | | 7.Medium only | 40 | 40 | | ![](https://static.igem.org/mediawiki/2018/6/60/T--Botchan_Lab_Tokyo--K0928_4_4.png) Indophenol(48h)MSX-4 | Sample Name | Phenol/EtOH(&microl) | Sodium nitroprusside/H2O(&microl) | Sodium hypochlorite(&microl) | | --- | --- | --- | --- | | 1.*E.coli*(pHY*nifB*~*hesA*+pMW*nifVSU*) MSX | 40 | 40 | 100 | | 2.*E.coli*(pHY+pMW)MSX | 40 | 40 | 100 | ![](https://static.igem.org/mediawiki/2018/1/12/T--Botchan_Lab_Tokyo--K092848M_4.png) 10/04 Detection of glutamate ![](https://static.igem.org/mediawiki/2018/4/41/T--Botchan_Lab_Tokyo--takumi18.png) 10/8transformation Sample 1.pMW*nifVSU*+*gluDH* 2.pHY*nifB*~*hesA*,pMW*nifVSU* +*gluDH* 3.pMW 10/9preculture Miniprep Sample:transformant(10/8) 10/11Miniprep Sample:Precultured *E.coli* Electrophoresis | Lane | sample/(µl) | | --- | --- | | 1.pMW*nifVSU**gluDH*(Extracted Plasmid) | 5 | | 2.pHY*nifB*~*hesA*,pMW*nifVSU**gluDH*(Extracted Plasmid) | 5 | ![](https://static.igem.org/mediawiki/2018/a/a7/T--Botchan_Lab_Tokyo--takumi12.png) Contact[![](https://static.igem.org/mediawiki/2018/f/f7/T--Botchan_Lab_Tokyo--facebook.jpg)](https://ja-jp.facebook.com/botchanlab/) [![](https://static.igem.org/mediawiki/2018/f/f8/T--Botchan_Lab_Tokyo--twitter.png)](https://twitter.com/BotchanLab) Sponsor[![](https://static.igem.org/mediawiki/2018/1/13/T--Botchan_Lab_Tokyo--sponsor--idt.jpg)](https://sg.idtdna.com/pages) [![](https://static.igem.org/mediawiki/2018/5/5e/T--Botchan_Lab_Tokyo--sponsor--promega.png)](https://www.promega.com/?cs=y) [1.Protocol](#link1) [2.Laboratory notebook](#link2)


[![](https://static.igem.org/mediawiki/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Botchan Lab Tokyo/Experiments

![](https://static.igem.org/mediawiki/2018/4/4f/2018_igem_mobile_menu_logo.svg)

![](https://static.igem.org/mediawiki/igem.org/3/3b/Menu_icon.svg)

[![](https://static.igem.org/mediawiki/2017/d/d8/T--Botchan_Lab_Tokyo--botchanlab_teamlogo.jpg)](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo)
[HOME](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo)

PROJECT

[RESULTS](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/Project)
[LABORATORY NOTEBOOK](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/Notebook)
[MODELING](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/Model)

LAB

[SAFETY](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/Safety)
[INTERLAB](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/InterLab)

PARTS

[COMPOSITE PARTS](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/Parts_Composite%20parts)
[PARTS BOOK](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/Parts_notebook)

HUMAN PRACTICE

[SILVER](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/HP/Silver)
[GOLD](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/Human_Practices)
[COLLABOLATIONS](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/Collaborations)

TEAM

[MEMBER](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/Team)
[ATTRIBUTION](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/Attributions)

# 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/2018/8/8b/HQ_page_logo.jpg)](https://2018.igem.org)

# Team:Botchan Lab Tokyo/Results

![](https://static.igem.org/mediawiki/2018/4/4f/2018_igem_mobile_menu_logo.svg)

![](https://static.igem.org/mediawiki/igem.org/3/3b/Menu_icon.svg)

[![](https://static.igem.org/mediawiki/2017/d/d8/T--Botchan_Lab_Tokyo--botchanlab_teamlogo.jpg)](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo)
[HOME](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo)

PROJECT

[RESULTS](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/Project)
[LABORATORY NOTEBOOK](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/Notebook)
[MODELING](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/Model)

LAB

[SAFETY](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/Safety)
[INTERLAB](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/InterLab)

PARTS

[COMPOSITE PARTS](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/Parts_Composite%20parts)
[PARTS BOOK](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/Parts_notebook)

HUMAN PRACTICE

[SILVER](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/HP/Silver)
[GOLD](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/Human_Practices)
[COLLABOLATIONS](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/Collaborations)

TEAM

[MEMBER](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/Team)
[ATTRIBUTION](https://2018.igem.org/Team%3ABotchan_Lab_Tokyo/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 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)