• 
• Home
• Project
  • Hunger
  • Hunger Modeling
  • Capture
  • Digestion
  • Digestion Modeling
• Consideration
  • Safety
  • Human Practice
• Method
  • Parts
  • Protocol
  • Material
• Team
• Notebook
  • Lab Work
  • Diary
• Acknowledgement
• Criteria

Lab Work

週ごとにページ作りました（by Kato）

• Week1
• Week2
• Week3
• Week4
• Week5
• Week6
• Week7
• Week8
• Week9

Week1: Monday 1st - Sunday 7th August

Monday
行った実験名：

使った試薬名、容量：

用いた機械：

行った人：

Tuesday

Wednesday

Thursday

Friday
DIGESTION:transformation of bellow parts.

4-17M:BBa_K325909(lux operon)
1-12M:BBa_E0240
2-17F:BBa_120260(low copy vector)
lactose promoter
double terminator

Saturday

Sunday

Week2: Monday 8th - Sunday 14th August

Monday

Tuesday

Wednesday

Thursday

Friday

DIGESTION:transformation of bellow parts.

4-17M:BBa_K325909(lux operon)
1-12M:BBa_E0240
2-17F:BBa_120260(low copy vector)
lactose promoter
double terminator

Saturday

Sunday

Week3: Monday 15th - Sunday 21th August

Monday

Tuesday

Wednesday

Thursday
Nutritional Signal(Sugiura,Shimosaka & Okumura)
PCR Amplification of glnL and glnG from gDNA of E.coli

--Primers--
glnL
Left primer: tctagaggagactgctttatggcaac
Right primer: actagtaggaactatcgtcatcgactac
glnG
Left primer: tctagaggtgacgtttatgcaacga
Right primer: actagtacacacaagctgtgaatcactc
annealing temperature was 55 degrees.

lane 1,2 &7,8: DNA ladder(λDNA digested Hindlll,100bp), lane 3,4: glnG1,glnG2, lane 5,6: glnL1,glnL2

After purification, the concentration of DNA are
glnG1: 127.8ng/ul
glnG2: 118.1ng/ul
glnL1: 137.4ng/ul
glnL2: 124.2ng/ul

Friday
Nutritional Signal(Shimosaka)
Restriction of glnG1 and glnG2
Cut them with Xbal and Spel for 2 hours at 37 degrees.
Then, gel extraction of digested.

Saturday

Sunday

Week4: Monday 22th - Sunday 28th August

Monday

Tuesday

Wednesday

Thursday
Luminescence(Kusaba, Terada, Hara):ハエの走行性実験①　♂、紫外線×2回、緑×2回　♀、紫外線×2回、緑×2回

Friday
Luminescence(Kusaba):ハエの走行性実験①　♂、赤外線×2回　♀、赤外線×2回

Nutritional Signal(Hashiya): Transformation of bellow parts.
4-17M:BBa_K325909(lux operon)
1-12M:BBa_E0240
2-17F:BBa_120260(low copy vector)

PCR amplification of

Saturday
Luminescence(Kusaba):ハエの走行性実験①　♂、赤×2回、青×2回　♀、赤×2回、青×2回

Sunday

Week5: Monday 29th August - Sunday 4th September

Monday

Tuesday
Nutritional Signal(Hashiya)
・PCR amplification of glnL and glnG from PCR products,glnL1 and glnG1.
　--Primers--
　glnL
　Left primer: ggaattcgcggccgcttctagaggagactgctttatggcaac
　Right primer: ggactagtaggaactatcgtcatcgactac
　glnG
　Left primer: ggaattcgcggccgcttctagaggtgacgtttatgcaacga
　Right primer: ggactagtacacacaagctgtgaatcactc
　annealing temperature was 55 degrees.

・PCR amplification of glnL+G and rpoN from gDNA of E.coli.
　--Primers--
　glnL+G
　Left primer: ggaattcgcggccgcttctagaggagactgctttatggcaac
　Right primer: ggactagtacacacaagctgtgaatcactc
　rpoN
　Left primer: ggaattcgcggccgcttctagaggttctgaacatgaagcaa
　Right primer: ggactagtatccttatcggttgggtca
　annealing temperature was 56 degrees.

　
　lane1: 100bp DNA ladder, lane2:glnL, lane3:glnG, lane4:glnG+L, lane5:rpoN from gDNA, lane6:rpoN from ASKA clone
　 　After purification, the concentration of DNA are
　glnL: 122.3 ng/ul
　glnG: 64.7 ng/ul
　glnL+G: 106.7 ng/ul
　rpoN from gDNA: 111.4 ng/ul

Wednesday
Nutritional Signal(Hashiya)
・Screening PCR of σ54 promoter + pSB1A3
　
　We cultured σ54 promoter5

Friday
Nutritional Signal(Hashiya)
・Restriction of σ54 promoter5,glnL, glnG, glnL+G and rpoN
　Cut them with EcoRl and Spel 　After purification, the concentration of DNA were
　σ54 promoter5: 23.6 ng/ul
　glnL: 28.1 ng/ul
　glnG: 26.3 ng/ul
　glnL+G: 15.3 ng/ul
　rpoN: 20.8 ng/ul

・Ligation reaction
　Ligated glnL, glnG and rpoN to pSB1K3.

Thursday
Nutritional Signal(Hashiya)
・Screening PCR of glnL, glnG, glnL+G and rpoN
glnL 　
glnG 　
glnL+G & rpoN 　
　We cultured glnL5, glnG4

Saturday
Nutritional Signal(Hashiya)
・Mini prep of glnL5 and glnG4
　glnL5: 43.9 ng/ul
　glnG4: 38.1 ng/ul

・Screening PCR of rpoN 　

Predation(Hashiya)
・PCR amplification of glmS
　--Primers--
　left primer:ggaattcgcggccgcttctagagcaggttgaccgacaacgata
　right primer:ggactagtacgcagggcatccatttat
　
　lane1: 100bp DNA ladder, lane2: glmS from gDNA, lane3: glmS from ASKA clone

・TA cloning of glmS
　Ligated glmS from gDNA to pTA vector.

Sunday
Nutritional Signal(Hashiya)
・Screening PCR of rpoN
　

・Transformation of bellow parts
　1-23L: BBa_B0015 (double terminator)
　1-18E: BBa_J23101 (constitutive promoter)
　1-18C: BBa_J23100 (constitutive promoter)

Week6: Monday 5th September - Sunday 11th September

Monday
Nutritional Signal(Hashiya)
・Screening PCR of glnL+G+double terminator

Tuesday
Luminescence(Kusaba, Hara):ハエの走行性実験②（②は改良版）　♂、緑×2回　♀、青×1回

Wednesday
Luminescence(Hara):ハエの走行性実験②　♂、青×2回　♀、緑×2回、青×1回

Thursday
Luminescence(Hara):ハエの走行性実験②　♂、紫外線×3回　♀、紫外線×3回

Friday

Saturday
Luminescence(Kusaba, Hara):ハエの走行性実験②　♂、青×2回、赤×2回　♀、青×2回、赤×2回　

Sunday
Luminescence(Kusaba, Hara):ハエの走行性実験②　♂、紫外線×2回、

Week7: Monday 12th September - Sunday 18th September

Monday

Luminescence:大腸菌の形質転換(Hashiya)　ハエの走行性実験②(Kusaba, Hara)

Tuesday

Luminescence:大腸菌はじめて光る。しかし光量は少ない。

Wednesday

Thursday

Friday

Digestion(Kajita)

PCR amplification of SAM-P20 and ChiA

We performed colony direct PCRs from a S. albogriseolus colony and a S. avermitilis colony.

Reaction mixture

Component	Volume(μl)
2x Buffer	25
2mM dNTPs	10
Primer 1	1.5
Primer 2	1.5
Template	X
KOD FX	1
ddH2O	up to 50

PCR condition

Predenature	94C	2m
Denature	98C	10s	30cycles
Annealing	56C	30s
Extension	68C	1m30s

We prepared two kinds of templates:

1. Picked a colony, suspended in 50ul of water and incubated 1min at 95 degree. Added 1ul to the reaction mixture.
2. Picked a colony and dipped in the reaction mixture.

Gel electrophoresis indicated that the PCR amplifications were successful for a sample, ChiA, but it was a faint band. We decided to retry direct PCR of SAM-P20 and PCR of ChiA.

Saturday

Digestion(Kajita)

Retry of PCR amplification of SAM-P20 and ChiA.

We amplified SAM-P20 by colony direct PCR and ChiA by PCR using the PCR product that performed yesterday.

Reaction mixture: The same components and volume as before.

PCR condition: The same PCR condition as before.

PCR for SAM-P20

We prepared two kinds of templates:

1. Picked a colony, suspended in 50ul of TE buffer (pH8.0) and incubated 1min at 95 degrees. Added 5ul to the reaction mixture.
2. Picked a colony and dipped in the reaction mixture.

PCR for ChiA

1μl of PCR product was added to the reaction mixture as template.

Gel electrophoresis indicated that the PCR amplifications were successful for all samples. However, an unexpected faint band, about 1000bp, was also observed in the sample of ChiA.

PCR purification of SAM-P20 and gel extraction of ChiA.

SAM-P20: 43.9 ng/μl

ChiA: 30.0 ng/μl

Restriction enzyme digestion

We performed restriction digestions for:

1. SAM-P20 with EcoRI and SpeI
2. ChiA with EcoRI and SpeI

Incubated overnight at 37 degrees.

Sunday

Digestion (Kajita)

Purification of digested products

SAM-P20 : 10.2 ng/μl

ChiA : 22.5 ng/μl

Ligation

Name	Vector	Insert
1	pSB1C3	SAM-P20
2	pSB1C3	ChiA
3	BBa_B0015	SAM-P20
4	BBa_B0015	ChiA

Incubated overnight at 16 degrees.

PCR amplification of BBa_R0011, lactose promoter

We done the amplification of lactose promoter. After that, we digested the product with DpnI, incubated 1 hour at 37 degrees.

Gel electrophoresis indicated that the PCR amplification was successful, but DpnI didn't work at all. So, we done the gel extraction.

Restriction enzyme digestion for pSB4K5 with EcoRI and PstI

Week8: Monday 19th September - Sunday 25th September

Monday

Tuesday

Wednesday

Thursday

Friday

Saturday

Sunday

Week9: Monday 26th September - Sunday 2nd October

Monday

Tuesday

Wednesday

Thursday

Friday

Saturday

Sunday