iGEM 2012 — Team UT-Tokyo

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# Team:UT-Tokyo/Notebook

### From 2012.igem.org

[![iGEM UT-Tokyo](https://static.igem.org/mediawiki/2012/8/82/UT-Tokyo_logo01.png)](https://2012.igem.org/Team%3AUT-Tokyo)

* [Home](https://2012.igem.org/Team%3AUT-Tokyo)

  + [Sweetaholic Energy Generator:
    Hydrogen Production from Sugar-rich Waste by E.coli](https://2012.igem.org/Team%3AUT-Tokyo)
* [Team](https://2012.igem.org/Team%3AUT-Tokyo/Team)

  + [Members](https://2012.igem.org/Team%3AUT-Tokyo/Team/Members)
  + [Collaboration](https://2012.igem.org/Team%3AUT-Tokyo/Team/Collaboration)
  + [Achievement](https://2012.igem.org/Team%3AUT-Tokyo/Team/Achievement)
  + [Acknowledgment](https://2012.igem.org/Team%3AUT-Tokyo/Team/Acknowledgment)
* [H2 E.coli](https://2012.igem.org/Team%3AUT-Tokyo/Project/H2_E.coli)

  + [Project1 H2 E.coli:](https://2012.igem.org/Team%3AUT-Tokyo/Project/H2_E.coli)
  + [Background](https://2012.igem.org/Team%3AUT-Tokyo/Project/H2_E.coli/Background)
  + [System](https://2012.igem.org/Team%3AUT-Tokyo/Project/H2_E.coli/System)
  + [Results](https://2012.igem.org/Team%3AUT-Tokyo/Project/H2_E.coli/Results)
  + [Discussion](https://2012.igem.org/Team%3AUT-Tokyo/Project/H2_E.coli/Discussion)
* [Inhibition without Knockout](https://2012.igem.org/Team%3AUT-Tokyo/Project/Inhibition)

  + [Project2 Inhibition without Knockout:](https://2012.igem.org/Team%3AUT-Tokyo/Project/Inhibition)+ [Background & System](https://2012.igem.org/Team%3AUT-Tokyo/Project/Inhibition/System)
    + [Results & Discussion](https://2012.igem.org/Team%3AUT-Tokyo/Project/Inhibition/Discussion)
* [Parts](https://2012.igem.org/Team%3AUT-Tokyo/Parts)

  + [The BioBrick Parts We Made](https://2012.igem.org/Team%3AUT-Tokyo/Parts)
* [Lab Work](https://2012.igem.org/Team%3AUT-Tokyo/LabWork)

  + [Regular Methods](https://2012.igem.org/Team%3AUT-Tokyo/LabWork/RegularMethods)
  + [Assay Methods](https://2012.igem.org/Team%3AUT-Tokyo/LabWork/AssayMethods)
  + [Lab Note](https://2012.igem.org/Team%3AUT-Tokyo/Notebook)
  + [Safety](https://2012.igem.org/Team%3AUT-Tokyo/Safety)
* [Human Practice](https://2012.igem.org/Team%3AUT-Tokyo/HumanPractice)

  + [Human Practice: Book Review](https://2012.igem.org/Team%3AUT-Tokyo/HumanPractice)
  + [Campus Festival](https://2012.igem.org/Team%3AUT-Tokyo/HumanPractice/CampusFestival)

# Notebook

![box-background image](https://static.igem.org/mediawiki/2012/8/8a/UT-Tokyo_LN-02.jpg)

## Correspondence table

| No. | Biobrick Parts |
| --- | --- |
| #1 | pConst |
| #2 | Argbox |
| #3 | RBS |
| #4 | Argbox×8 |
| #5 | d.term |
| #6 | fake of fhlA |
| #7 | fhlA(P-siteless) |
| #8 | hycAp |
| #9 | Ptrc-RBS-rocF |
| #10 | fhlA(E363G) |
| #11 | RFP(psb1K3) |
| #20 | pLac |
| #40 | RBS(weak)-GFP-d.term |
| #44 | RBS(strong)-GFP-d.term |
| #66 | RFP(psb1C3) |

## Lab Notes

## May

### 1st week: 5/27-6/2

#### May 29th

* Making LB medium

#### May 30th

* Making LB Agar
* Making LB Amp Plates

#### May 31st

* Preculture

## June

### 2nd week: 6/3-6/9

#### June 5th

* Plating JM109

#### June 6th

* preculture

#### June 7th

* Measuring OD by spectroscope
* Meking electrocompetent cells
* Electroporating of a test plasmid

#### June 8th

* Caluculatiing transformation efficiency

### 3rd week: 6/10-6/16

none

### 4th week: 6/17-6/23

none

### 5th week: 6/24-6/30

#### June 26th

* Making LB tetracycline plate

#### June 27th

#### June 28th

* Making LB kanamycin plate and chloramphenicol plate
* Transformation with a test plasmid

#### June 29th

* Transformation with #1(constitutive promoter) and #11(RBS)

## July

### 6th week: 7/1-7/7

#### July 3rd

* Making K solution
* Colony PCR DNA fragment assumed as fhlA (#6) for cloning

#### July 4th

* Miniprep #1, #11

#### July 5th

* Making 1% agarose solution
* Electrophoresis (#6)
* Digest (#1\_EP, #11-\_EP)

#### July 6th

* Colony PCR (#6)

### 7th week: 7/8-7/14

#### July 9th

* Electrophoresis (#6)

#### July 10th

* Transformation
  + #3(RBS medium), #5(d.term), #20(pLac)

#### July 11th

* Preculture
  + #3, #5, #20
* Gel Extraction(#6)

#### July 12th

* The continuation of Gel Extraction(#6)
* Miniprep
  + #3, #5, #20

#### July 13th

* Nanodrop (#6).
* Digestion (#3:E-P, #5:E-P, #20:E-P)
* Electrophoresis (#3, #5, #20)

#### July 14th

* Miniprep (#1,3,5,11,20)
* Gel extraction (#6)

### 8th week: 7/15-7/21

#### July 17th

* Transformation (#1A2,#3,#5,#20)
* PCR (parts of 1. and #3,#20(Colony PCR))
* Incubate #5.

#### July 18th

* Miniprep (#1A2,#3,#5,#20)
* PCR using u.p (#1A2,#3,#5,#20)
* Colony PCR (#3,#20,H₂O)
* Electrophoresis (#1A2,#3,#5,#20, #3,#20, H₂O)
* PCR (2011’s #1,#36 and 2012’s #3,#5,#20, H₂O)

#### July 20th

* Miniprep.
* Electrophoresis (7/19’s PCR product(#1,#3,#5,#20,#36) )
* Colony PCR (#3,#5,#20)

#### July 21st

* Electrophoresis (7/20’s PCR product (#3,#5,#20))

### 9th week: 7/22-7/28

#### July 24th

* Making agar medium plate contain ampicillin
* PCR (#6, #8)
* Electrophoresis (#6, #8)
* Transformation (#1)
* Culture (#5, #20)

#### July 25th

* Miniprep (#5, #20)
* Purify (#6, #8)
* Digestion (#3:S-P,#5:E-X,#11:E-S,#20:S-P)
* Electrophoresis (#5, #11)
* Ligation (#5,#11) (?)

#### July 26th

* Culture (#1)
* Digestion (#3:S-P, #5:E-X, #11:E-S, #20:S-P, fhlA:E-S, hycA:E-S)
* Electrophoresis (#3:S-P, #5:E-X, #11:E-S, #20:S-P, fhlA:E-S, hycA:E-S)
* Gel extraction (#3:S-P, #5:E-X, #11:E-S, #20:S-P, fhlA:E-S, hycA:E-S)

#### July 27th

* Culture, Miniprep (#1)
* Digestion (#5-A:E-X, #11-A:X-P)
* Electrophoresis (#3:S-P, #20:S-P, #11:E-S)
* Gel extraction (#3:S-P, #20:S-P, #11:E-S, E-S vector, hycAp:E-S)

#### July 28th

* Digestion(#1,3,5,11,hycAp)
* Gel extraction(#1,3,5,11,hycAp)
* Cloning(fhlA)

### 10th week: 7/29-8/4

#### July 30th

* Digestion (#3:S-P, #11:X-P)
* Electrophoresis (#3, #11)
* Gel extraction (#3, #11)
* Ligation (#3, #11)
* Transformation (ligation product; #3-11)

#### August 1st

* Digestion (#3:S-P, #11:X-P)
* Electrophoresis (#3, #11)
* Gel extraction (#3, #11)
* Ligation (#3, #11)
* Transformation (ligation product; #3-11)
* Miniprep (#3, #5, #11)

#### August 2nd

* Colony PCR (#3-11)
* Culture (#3-11)
* Colony pick up, culture (red colony of #11)
* Digestion (#3:S-P, #5:E-X)

#### August 3th

* Gel extraction (#3:S-P, #5:E-X)
* Making solution of Kanamycin (100mg/mL)
* Culture, miniprep (#11)
* Culture (#11)

#### August 4th

* Plating, Miniprep (#11)

## August

### 11th week: 8/5-8/11

#### August 6th

* Culture, Colony PCR (#3-11)

#### August 7th

* Colony PCR, culture (#3-11)
* Miniprep, Digestion, Gel extraction (culture product; #3-11)

#### August 8th

* Digestion (#5: E-X)
* Gel extraction (#5)
* Miniprep (#5)
* Colony PCR (culture product; #3-11)

#### August 9th

* Colony PCR (#3-11)
* Culture, Miniprep (#11)
* Digestion (#11:X-P)
* Cloning (#6,#8)
* Ligation
* Gel extraction (ligation product; #5, #11)
* Ligation, Transformation (#3, #11)

#### August 10th

* Colony PCR (#3-11)
* Miniprep (#3)
* Digestion (#3:S-P)
* Cloning, Colony PCR (#6,#8)
* cut check, PCR check (#3-11)
* Electrophoresis (ligation product; #3, #11, PCR product; #6)
* Gel extraction (#6)

#### August 11th

* electrophoresis(PCR product; #3-11)
* Digestion (#6:E-S)
* electrophoresis (#8)

### 12th week: 8/12-8/18

#### August 13th

* Check, Culture (#3-11)
* Transformation (test plasmid)

#### August 15th

* Gel extraction (cut check product; #3, #11)
* Ligation, Transformation (#3-#11)
* Make Vector for #6
* Digestion (#3: E-S)
* Gel extraction (digestion product; #3, #6(8/11)
* PCR check (ligation product; #3-11)
* Cloning (#6)

#### August 16th

* Gel extraction (#6)
* cloning (#6)
* PCR check (#3-11)
* PCR-cleanup (#8)
* culture (#3-11)

#### August 17th

* electrophoresis (#6)
* Miniprep (#3-11)
* Digestion (#3-#11: E-S, #6: E-S)
* Gel extraction (#3-11)
* Ligation, Transformation (#3-11-5)
* Making solution of Ampicillin

### 13th week: 8/19-8/25

#### August 20th

* PCR check (#3-11-5)
* Digestion (#8: E-S)
* Cloning (#6)
* Digeston (#8: E-S)
* Gel extraction (#6)

#### August 21th

* Colony PCR (#1)
* Digestion (#6: E-S, #3-11: E-S, #20: S-P)
* Ligation (#3-11, #5)
* Gel extraction (#3-11, #6)
* Ligation (#6-pSB1A2)
* Electrophresis (#3, #5; made previously)

#### August 22th

* Cloning (#6; from test plasmid)
* Colony PCR (#3-11-5(colony is not red), #20, 8/21 ligation product;#6, #8)
* Miniprep,Frozen stock (culture product; #1, #5, #20)
* Transformation (#8, #3-11-5)
* Digestion (#6:X-S)
* Making solution of Ampicillin
* cloning retry (#6)

#### August 23th

* PCR check (ligation product; #6)
* Cloning (#6)

Colony PCR (#1, #6, #8, #11, #3-11-5, #3-11)

#### August 24th

* Digestion (#6, E-S, #3-11, E-S)
* Colony PCR #3-11
* Electrophresis, Gel extraction (#6)
* Vector insert (#6)
* Sequencing (#8)

#### August 25th

* Digestion (#6: E-S)
* Colony PCR (#6-Vector)
* Gel extraction (#3-11)

### 14th week: 8/26-9/2

#### August 26th

* Electrophresis (#6)
* Master plate (#6)
* Colony PCR (#3-11-5)
* Preculture (#6)

#### August 27th

* Ligation (Miniprep product; #3-11, #5)
* Digestion retry (#3-11, #5)
* Miniprep (#6)
* Mutagenesis (#6)
* Sequencing (#6)

#### August 28th

* Mutagenesis(#6)
* Digestion(#3-11,#5)

#### August 29th

* Mutagenesis by inverse PCR(#6)
* Digestion(#5)
* Cut check(#6)
* PCR check(#6)
* Sequencing(#6)
* Making agar medium plate contain Chloramphenicol

#### August 30th

* Digestion(#5,#3-11,#8)
* Sequencing(#6)
* Mutagenesis(#6)

#### August 31st

* Colony PCR(#6)
* Digestion(#3-11,#5)
* Ligation(#3-11+#5)
* Transformation(#3-11-5,#66)
* Culture(#66)

#### September 1st

* Cloning(#6)
* Miniprep(#66)

## September

### 15th week: 9/3-9/9

#### September 3rd

* Miniprep(#3,#11)
* Colony PCR(#3-11-5)
* Transformation(#2)

#### September 4th

* Digestion(#3,#6,#11)
* Ligation(#3,#6)
* Miniprep(#2)

#### September 5th

* Ligation(#6+1A2)
* Check PCR(#6)
* Digestion(#2,8)

#### September 6th

* Mutagenesis(#7)
* Sequencing(#6)
* Digestion(#5,#66)
* Transformation(#2,#6)

#### September 7th

* Colony PCR(#2,#8)
* Cut check(EX,ES,XP,SPcut)
* Digestion(#5,#66)
* Ligation(#66+#5)
* Transformation(#66-5)
* Mutagenesis(#7)

#### September 8th

* Sequencing(#6)
* Colony PCR(#66-5)
* Miniprep(#2)

### 16th week: 9/10-9/16

#### September 10th

* Mutagenesis(#7)
* Digestion(#2)
* Transformation(#9,#40)

#### September 11th

* Transformation(#44)
* Mutagenesis(#7)
* Miniprep(#6)
* Colony PCR(#7)

#### September 12th

* Culture(#44)
* Miniprep(#44)
* Mutagenesis(#7)
* Transformation(#7)

#### September 13th

* Culture(#1,#7,#20)
* Colony PCR(#7)
* Mutagenesis(#7)

#### September 14th

* Inverse PCR(#10)
* Digestion(#3,#7)
* Ligation(#3-7)
* Transformation(#3-7,#10)

#### September 15th

* Digestion(#1,#7,#8,#44,#66)
* Sequencing(#7,#10)
* Colony PCR(#44,#3-7)

#### September 16th

* Digestion(#1,#3,#7-5,#8,#10,#44,#66)
* Transformation(#3-7-5,#3-10)

### 17th week: 9/17-9/23

#### September 17th

* Miniprep(#7-5,#44)
* Digetion(#3,5,10)
* Transformation(#3-7-5,3-10-5)

#### September 18th

* Miniprep(#7-5,#44)
* Digetion(#3,#5,#10)
* Transformation(#3-7-5,#3-10-5)

#### September 19th

* Transformation(#4)
* Miniprep(#3-7-5,#3-10-5)
* Digestion(#3-7-5,#3-10-5,#20,#66)

#### September 20th

* Miniprep(#4)
* Digestion(#3-7-5,#3-10-5,#4,#20,#66)
* Transformation(#20-3-7-5,#20-3-10-5,#4-4)

#### September 21st

* Digestion(#3-7-5,#3-10-5,#4,#20,#66)
* Transformation(#20-3-7-5,#20-3-10-5,#4-4,#7+1C3,#10+1C3)
* Gas chromatography (preliminary experiment using WT)

#### September 22nd

* Digestion(#3-7-5,#3-10-5,#4,#20,#66)
* Transformation(#20-3-7-5,#20-3-10-5,#4-4)
* Miniprep(#7+1C3,#10+1C3)

#### September 23rd

* Digestion(#3-7-5,#3-10-5,#4,#8,#20,#66)
* Transformation(#20-3-7-5,#20-3-10-5,#4-4)
* Colony PCR(#20-3-7-5)

### 18th week: 9/24-9/26

#### September 24th

* Digestion(#3-7-5,#3-10-5,#4,#8,#20,#66)
* Transformation(#20-3-7-5,#20-3-10-5,#4-4)
* Colony PCR(#20-3-7-5,#4+1C3)

#### September 25th

* Digestion(#3-7-5,#3-10-5,#4,#20,#66)
* Transformation(#20-3-7-5,#20-3-10-5,#4-4)
* Colony PCR(#8-44)

Gas chromatography (WT,#6,#10)

#### September 26th

* Digestion(#20-3-7-5,#20-3-10-5,#4,#66,#8-44)
* Transformation(#8-44-20-3-7-5-1C3,#8-44-20-3-10-5-1C3)
* Culture(4-4-1C3)
* Gas chromatography (preparation of analytical line,WT,#7,#10)

![logo](https://static.igem.org/mediawiki/2012/5/5f/UT-Tokyo_cakeH2.png)

[Follow @UT\_Tokyo](https://twitter.com/UT_Tokyo)

[May](#May)

[June](#June)

[July](#July)

[August](#August)

[September](#September)

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[Assay Methods](https://2012.igem.org/Team%3AUT-Tokyo/LabWork/AssayMethods)

[Safety](https://2012.igem.org/Team%3AUT-Tokyo/Safety)
>>

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