Team:Kyoto/Lab Work

From 2011.igem.org

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Digestion (Mori, Nobeyama)
Digestion (Mori, Nobeyama)
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Colony PCR
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Revision as of 22:18, 4 October 2011

Contents

Lab Work

Week1: Monday 1st - Sunday 7th August

Monday

Tuesday

Wednesday

Thursday

Friday

Saturday

Sunday

Week2: Monday 8th - Sunday 14th August

Monday

Tuesday

Wednesday

Thursday


Friday
12th

Digestion:transformation of parts listed as below.

•lactose promoter(ampicilin)
•double terminator(ampicilin)
•4-17M:BBa_K325909(chloramphenicol)
•1-12M:BBa_E0240(ampicilin)
•2-17F:BBa_120260(kanamycin)

After transformation, we put E.coli in the plate which is containing the each antibiotic.

We failed to conduct transformation of 4-17M,1-12M,and2-17F ,but transformation of lactose promoter and double terminator worked out.


Saturday

Sunday

Week3: Monday 15th - Sunday 21th August

Monday

Tuesday

Wednesday

Thursday

Digestion:retry of transformation

We could not work out transformation of 4-17M,1-12M,and2-17F. This was why we tried again.
However, these experiments were failed.

Digestion:liquid culture of lactose promoter and double terminator

Picked colonies by using tips ,then put in the LB medium(LB 3ml ampicilin 3μl)

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.
Kyoto-Gel0818.jpg
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.

Digestion:Mini prep of lactose promoter and double terminator.

This is results of the experiment.
•lactose promoter 74.3μl/ml
•double terminator 6.7μl/ml
lactose promoter expressed GFD,so we judged this part was not correct one.

Digestion:Transformation of parts listed as below

4-17M(luciferage)
1-12M(GFP-dT)
2-17F(middle copy vector:iGEM 2009kit)

these transformation were failed.

Saturday

Sunday

Week4: Monday 22th - Sunday 28th August

Monday

Digestion:Transformation of these parts as below

4-17M:BBa_k325909(luciferage)
1-12M:BBa_E0249(GFP-dT)
2-17F:BBa_I20260(middle copy vector:iGEM 2010kit)
these transformation were failed.

Tuesday

Digestion:Transformation

•1-5E(pSB3K3)

Wednesday

Digestion:DNeasy of these parts described as below

•JCM4616 16.9μg/ml 1.48 260/280
•JCM5070 7.7μg/ml 1.40 260/280

Digestion:liquid culture

•double terminator
•1-5E

Thursday

Digestion:Restriction enzyme digestion

•double terminator(77.4μg/ml)
result

this experiment was failed.
the reason of this failure was

Luminescence(Kusaba, Terada, Hara): the experiment about flies' phototaxis ① ♂:UV×2, green×2, ♀:UV×2, green×2

Friday

Luminescence(Kusaba):the experiment about flies' phototaxis ① ♂:IR×2 ♀:IR×2

Digestion:Miniprep

•1-5E 7.1μg/ml 1.53 260/280

Digestion:Transformation of a part described as below.

•1-6G:BBa_R011(pSB1A2)

Digestion:DNeasy of these parts described as below.

•JCM4616 4.4μg/ml 1.22 260/280 0.28 260/230
•JCM5070 3.2μg/ml 1.38 260/280 0.25 260/230

Digestion:electrophoresis

•double terminator(77.4μg/ml)
•1-5E(7.1μg/ml)
results

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):the experiment about flies' phototaxis ① ♂:red×2, blue×2 ♀:red×2, blue×2

Sunday

Week5: Monday 29th August - Sunday 4th September

Monday

Tuesday

Digestion: Transformation of 1-5E
Digestion: liquid culture of JCM5070 and JCM4616

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.

 Kyoto-Gel08300.jpg
 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

Digestion(nobeyama izumi komatsu):DNeasy of JCM5070 and JCM4616
these results were described as below
JCM5070-1 5.2μg/ml
JCM5070-2 4.9μg/ml
JCM5070-3 4.4.μg/ml
JCM4616-1 10.7μg/ml
JCM4616-2 10.0μg/ml
JCM4616-3 16.2μg/ml

Digestion:liquid culture of 1-6E,JCM5070,and JCM4616

Nutritional Signal(Hashiya)
・Screening PCR of σ54 promoter + pSB1A3
 Kyoto-Gel08301.jpg
 We cultured σ54 promoter5

Friday

Digestion: Mini prep of 1-6E-1,1-6E-2,and1-6E-3.

RESULTS
1-6G-1 there were no plasmid.
1-6G-2 there were no plasmid.
1-6G-3 132.3μg/ml 1.77 260/280 2.16 260/230

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

Digestion:Mini prep of 1-6E,σ54-5,and σ54-10

these results were described as below
1-6E-1 failure
1-6E-2 failure
1-6E-3 failure
σ-54-5 26.4μg/ml 1.70μg/ml 260/280 1.60μg/ml 260/230
σ-54-10 failure

Digestion:DNeasy of JCM5070 and JCM 4616.

JCM5070W1  7.1μg/ml
JCM5070W2  13.6μg/ml
JCM5070G1  8.1μg/ml
JCM5070G2  4.8μg/ml
JCM4616W1  64.9μg/ml
JCM4616W2  7.5μg/ml
JCM4616G1  10.1μg/ml
JCM4616G2   8.1μg/ml

Nutritional Signal(Hashiya)
・Screening PCR of glnL, glnG, glnL+G and rpoN

glnL Kyoto-Gel09020.jpg
glnG Kyoto-Gel09021.jpg
glnL+G & rpoN Kyoto-Gel09022.jpg

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  Kyoto-Gel09020.jpg

Predation(Hashiya)
・PCR amplification of glmS
 --Primers--
 left primer:ggaattcgcggccgcttctagagcaggttgaccgacaacgata
 right primer:ggactagtacgcagggcatccatttat
 Kyoto-Gel09030.jpg
 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
 Kyoto-Gel09020.jpg

・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

Digestion;retry of liquid culture of 1-6G-1 and 1-6G-2

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

Tuesday

Digestion: Miniprep of 1-6G which was cultured on September 5

these results were described as below.
1-6G-1 139.1μg/ml 1.63 260/280 2.10 260/230
1-6G-2 130.3μg/ml 1.75 260/280 2.19 260/230

Digestion:ethanol precipitation of JCM5070W2(1st September)and JCM4616-3(31th August).

these results were described as below.
•JCM5070W2 37.2μg/ml 1.20 260/280 0.65 260/230
•JCM4616-3 53.0μg/ml 1.13 260/280 0.58 260/230

Luminescence(Kusaba, Hara):the experiment about flies' phototaxis ②(② is the improved version)♂:green×2 ♀:blue×1

Wednesday

Luminescence(Hara):the experiment about flies' phototaxis ② ♂:blue×2, green×2, ♀:blue×2, green×2

Thursday

Luminescence(Hara):the experiment about flies' phototaxis ② ♂:UV×2 ♀:UV×2

Friday

Saturday

Luminescence(Kusaba, Hara):the experiment about flies' phototaxis ② ♂:blue×2, green×2, red×2, IR×2, ♀:blue×2, green×2, red×2, IR×2 

Sunday

Luminescence(Kusaba, Hara):the experiment about flies' phototaxis ② ♂:UV×2

Week7: Monday 12th September - Sunday 18th September

Monday

Luminescence:transformation of E.coli(Hashiya)
the experiment about flies' phototaxis ②(Kusaba, Hara) ♀:blue×4, UV×2, red×4, IR×4, ♂:blue×2, red×4, IR×2

Tuesday

Luminescence:E.coli emitted light for the first time. But the amount of light is little.

Wednesday

Thursday

Friday

Digestion(Mori)

PCR amplification of SAM-P20 and ChiA

We performed colony direct PCRs from a S. albogriseolus colony and a S. avermitilis colony.
Reaction mixture
ComponentVolume(μl)
2x Buffer25
2mM dNTPs10
Primer 11.5
Primer 21.5
TemplateX
KOD FX1
ddH2Oup to 50
PCR condition
Predenature94C2m
Denature98C10s30cycles
Annealing56C30s
Extension68C1m30s
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(Mori)

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 (Mori)

Purification of digested products

SAM-P20 : 10.2 ng/μl
ChiA : 22.5 ng/μl


Ligation

NameVectorInsert
1pSB1C3SAM-P20
2pSB1C3ChiA
3BBa_B0015SAM-P20
4BBa_B0015ChiA
Incubated overnight at 16 degrees.

Week8: Monday 19th September - Sunday 25th September

Monday

Tuesday

Digestion (Mori)

Transformation

NameVectorInsertGrowth
1pSB1C3SAM-P20No
2pSB1C3ChiAYes
3BBa_B0015SAM-P20Yes
4BBa_B0015ChiAYes

Wednesday

Digestion (Mori, Nobeyama)

Colony PCR

Thursday

Friday

Saturday

Sunday
Luminescence(Kusaba, Hara):the experiment about flies' phototaxis ② ♂:E.coli×4 ♀:E.coli×4

Digestion (Mori)

Measurement assay for chitinase A1.

We performed standard measurement and trial of sample measurement.
Kyoto standardforchitinase.png

Week9: Monday 26th September - Sunday 2nd October

Monday
Luminescence(Kusaba, Hara):the experiment about flies' phototaxis ② ♂:E.coli×4, ♀:E.coli×4

Tuesday

Wednesday

Thursday

Friday

Saturday

Sunday