Team:Kyoto/Lab Work

From 2011.igem.org

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<html><a name="lab-week1"></a></html>
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== Week1: Monday 1st - Sunday 7th August ==
== Week1: Monday 1st - Sunday 7th August ==
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<html><a name="lab-week2"></a></html>
<html><a name="lab-week2"></a></html>
== Week2: Monday 8th - Sunday 14th August ==
== Week2: Monday 8th - Sunday 14th August ==
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'''Friday'''<br/>
12th <br/>
12th <br/>
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</div>
</div>
      
      
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'''Sunday'''<br/>
 
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<html><a name="lab-week3"></a></html>
<html><a name="lab-week3"></a></html>
== Week3: Monday 15th - Sunday 21th August ==
== Week3: Monday 15th - Sunday 21th August ==
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'''Monday'''<br/>
 
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'''Tuesday'''<br/>
 
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'''Thursday'''<br/>
<div class="digestion">
<div class="digestion">
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these transformation were failed.
these transformation were failed.
</div>
</div>
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'''Saturday'''<br/>
 
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'''Sunday'''<br/>
 
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<html><a name="lab-week4"></a></html>
<html><a name="lab-week4"></a></html>
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== Week5: Monday 29th August - Sunday 4th September ==
== Week5: Monday 29th August - Sunday 4th September ==
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'''Monday'''<br/>
 
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'''Tuesday'''<br/><br/>
'''Tuesday'''<br/><br/>
<div class="digestion">
<div class="digestion">
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・Screening PCR of glnL+G(4. sept)<br/>
・Screening PCR of glnL+G(4. sept)<br/>
[[File:kyoto-gel-09060.jpeg]]<br/>
[[File:kyoto-gel-09060.jpeg]]<br/>
 +
　We cultured glnL+G-9.<br/>
・Ligation<br/>
・Ligation<br/>
　We ligated rpoN to pSB1C3.<br/>
　We ligated rpoN to pSB1C3.<br/>
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<div class="capture">
<div class="capture">
Capture(Hara):the experiment about flies' phototaxis ②　♂:blue×2, green×2, ♀:blue×2, green×2
Capture(Hara):the experiment about flies' phototaxis ②　♂:blue×2, green×2, ♀:blue×2, green×2
 +
</div>
 +
<div class="nutrition">
 +
Nutritional Signal(Hashiya)<br/>
 +
・Mini prep of glnL+G-9<br/>
 +
　The concentration of DNA was 70.4ng/ul.<br/>
 +
・Restriction of double terminator and glnL+G-9<br/>
 +
　Cut double terminator with EcoRl and Xbal.<br/>
 +
　Cut glnL+G-9 with EcoRl and Spel.<br/>
 +
[[File:kyoto-gel-09070.jpeg]]<br/>
 +
　lane1:λ marker, lane2&3:glnL+G cut with E&S, lane4:double terminator cut with E&X, lane5&6:pSB1A3 cut with E&S<br/>
</div>
</div>
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<div class="capture">
<div class="capture">
Capture(Hara):the experiment about flies' phototaxis ②　♂:UV×2 ♀:UV×2
Capture(Hara):the experiment about flies' phototaxis ②　♂:UV×2 ♀:UV×2
 +
</div>
 +
<div class="nutrition">
 +
Nutritional Signal(Hashiya)<br/>
 +
・Screening PCR of rpoN<br/>
 +
[[File:kyoto-gel-09071.jpeg]]<br/>
 +
　We cultured rpoN-9 and rpoN-11<br/>
</div>
</div>
 +
 +
'''Thursday'''<br/>
 +
<div class="capture">
 +
Capture(Hara):the experiment about flies' phototaxis ②　♂:UV×2 ♀:UV×2
 +
</div>
 +
<div class="nutrition">
 +
Nutritional Signal(Hashiya)<br/>
 +
・Sequence<br/>
 +
　We tryed to get sequence of sigma54 promoter-5, glnL-5, glnG-4, rpoN-9 and glnL+G-9. However, they were too thin  to get sequence. So, we cultured all of them.<br/>
 +
</div>
 +
'''Friday'''<br/>
'''Friday'''<br/>
 +
<div class="nutrition">
 +
Nutritional Signal(Hashiya)<br/>
 +
・Restriction<br/>
 +
　Before getting sequence, we cut plasmid to know whether it is correct plasmid.<br/>
 +
　We cut rpoN-9 with Hindlll, BamHl & Pstl.<br/>
 +
　We also cut Lux operon with Hindll, EcoRl & Pstl.<br/>
 +
[[File:kyoto-gel09100.jpeg]]<br/>
 +
　lane1 & 2:1kb DNA ladder and 100bp DNA ladder, lane3,4:digested rpoN-9, lane5,6,7&8:digested lux operon<br/>
 +
　From this photo, we noticed lux operon were correct but rpoN-9 has some problem.<br/>
 +
</div>
 +
'''Saturday'''<br/>
'''Saturday'''<br/>
<div class="capture">
<div class="capture">
Capture(Kusaba, Hara):the experiment about flies' phototaxis ②　♂:blue×2, green×2, red×2, IR×2, ♀:blue×2, green×2, red×2, IR×2　
Capture(Kusaba, Hara):the experiment about flies' phototaxis ②　♂:blue×2, green×2, red×2, IR×2, ♀:blue×2, green×2, red×2, IR×2　
 +
</div>
 +
<div class="nutrition">
 +
Nutritional Signal(Hashiya)<br/>
 +
・Mini prep<br/>
 +
　The concentration of DNA were<br/>
 +
　sigma 54 promoter      135.7 ng/ul<br/>
 +
　glnL-5                143.2 ng/ul<br/>
 +
　glnG-4                126.2 ng/ul<br/>
 +
　glnL+G-9              25.7 ng/ul<br/>
 +
　rpoN-10                102.0 ng/ul<br/>
 +
　rpoN-11                95.1 ng/ul<br/>
 +
・Restriction<br/>
 +
　Before getting sequence, we cut plasmid to know whether it is correct plasmid.<br/>
 +
　We cut rpoN-9, rpoN-10 and rpoN-11 with EcoRl & Pstl.<br/>
 +
[[File:kyoto-gel09110.jpeg]]<br/>
 +
lane1&2:1kb DNA ladder and 100bp DNA ladder, lane5:digested rpoN-9, lane6:digested rpoN-10, lane7:digested rpoN-11<br/>
 +
　This photo shows rpoN-9 has some problem.<br/>
 +
・inverse PCR<br/>
 +
　To erase restriction site of Pstl in rpoN, we did inverse PCR.<br/>
 +
　Primer1:gcaagatgccaaatggttgatcaagagtc<br/>
 +
　Promer2:agattgctgcggataaactgg<br/>
</div>
</div>
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Capture:transformation of E.coli(Hashiya)<br>
Capture:transformation of E.coli(Hashiya)<br>
the experiment about flies' phototaxis ②(Kusaba, Hara) ♀:blue×4, UV×2, red×4, IR×4, ♂:blue×2, red×4, IR×2
the experiment about flies' phototaxis ②(Kusaba, Hara) ♀:blue×4, UV×2, red×4, IR×4, ♂:blue×2, red×4, IR×2
 +
</div>
 +
<div class="nutrition">
 +
Nutritional Signal(Hashiya)<br/>
 +
・Sequencing<br/>
 +
　We found glnG-4 is correct. The others, sigma 54 promoter, glnL-5 and glnL+G-9 are not correct.<br/>
</div>
</div>
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<div class="capture">
<div class="capture">
Capture:E.coli emitted light for the first time. But the amount of light is little.
Capture:E.coli emitted light for the first time. But the amount of light is little.
 +
</div>
 +
</div>
 +
<div class="nutrition">
 +
Nutritional Signal(Hashiya)<br/>
 +
・Screening PCR of glnL<br/>
 +
[[File:kyoto-gel0913.jpeg]]<br/>
 +
　We cultured lane2's strain as glnL-9.<br/>
</div>
</div>
'''Wednesday'''<br/>
'''Wednesday'''<br/>
 +
<div class="nutrition">
 +
Nutritional Signal(Hashiya)<br/>
 +
・Mini prep of glnL-9<br/>
 +
　The concentration of DNA was 136.1 ng/ul<br/>
 +
・Sequencing<br/>
 +
　We found glnL-9 is correct.<br/>
 +
</div>
'''Thursday'''<br/>
'''Thursday'''<br/>
 +
<div class="nutrition">
 +
Nutritional Signal(Hashiya)<br/>
 +
・PCR amplification of sigma 54 promoter<br/>
 +
　We remake sigma 54 promoter.<br/>
 +
　DNA oligo1:cggaattcgcggccgcttctagagattgca<br/>
 +
　DNA oligo2:gaatgttgcaccaatatagtgcttcaatggaaacattaagcaccatgttggtgcaatctctagaagcggc<br/>
 +
　DNA oligo3:gaatgttgcaccaatatagtgcttcaatggaaacattaagcaccatgttggtgcaatctctagaagcggc<br/>
 +
　DNA oligo4:ggactagtaaaagcgaaatctgtgccaacttttaaattgcccctaaaaggcgttatcatgcgcacc<br/>
 +
・Restriction of sigma 54 promoter.<br/>
 +
　We cut PCR product with EcoRl and Spel.<br/>
 +
・Gel extraction of sigma 54 promoter<br/>
 +
[[File:kyoto-gel0916.jpeg]]<br/>
 +
　After gel extraction, the concentration of DNA was 18.7 ng/ul.<br/>
 +
　The concentration of DNA was 136.1 ng/ul<br/>
 +
・Screening PCR of rpoN<br/>
 +
[[File:kyoto-gel09161.jpeg]]<br/>
 +
[[File:kyoto-gel09162.jpeg]]<br/>
 +
</div>
'''Friday'''<br/>
'''Friday'''<br/>
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: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.
: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.
 +
</div>
 +
 +
<div class="nutrition">
 +
Nutritional Signal(Hashiya)<br/>
 +
・Screening PCR of rpoNm<br/>
 +
　[[File:kyoto-gel0917.jpeg]]<br/>
 +
　[[File:kyoto-gel09171.jpeg]]<br/>
 +
　We cultured lane7's strain as rpoNm-11<br/>
</div>
</div>
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:Incubated overnight at 37 degrees.
:Incubated overnight at 37 degrees.
 +
 +
</div>
 +
<div class="nutrition">
 +
Nutritional Signal(Hashiya)<br/>
 +
・Restriction of rpoNm-11<br/>
 +
　We cut rpoNm-11 with EcoRl and Pstl.<br/>
 +
　[[File:kyoto-gel0918.jpeg]]<br/>
 +
　This photo shows the restriction site of rpoN were relieved.<br/>
</div>
</div>
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== Week8: Monday 19th September - Sunday 25th September ==
== Week8: Monday 19th September - Sunday 25th September ==
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'''Monday'''<br/>
 
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'''Tuesday'''<br/>
'''Tuesday'''<br/>
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</div>
</div>
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+
<div class="nutrition">
 +
Nutritional Signal(Hashiya)<br/>
 +
・Restriction of glnL and glnG<br/>
 +
　We cut glnL and glnG with EcoRl and Spel<br/>
 +
・Gel extraction of glnL and glnG<br/>
 +
[[File:kyoto-gel-0920.jpeg]]<br/>
 +
lane1:1kb DNA ladder, lane2&3:glnL, lane4&5:glnG<br/>
 +
　After gel extraction, the concentration of DNA were<br/>
 +
　glnL:18.7 ng/ul<br/>
 +
　glnG:10.0 ng/ul<br/>
 +
・Sequencing of rpoNm-11<br/>
 +
　We found rpoNm-11 is correct.<br/>
 +
</div>
'''Wednesday'''<br/>
'''Wednesday'''<br/>
<div class="digestion">
<div class="digestion">
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Gel electrophoresis shown that the ligation and transformation of pSB1C3 with ChiA was successful.
Gel electrophoresis shown that the ligation and transformation of pSB1C3 with ChiA was successful.
</div>
</div>
-
'''Thursday'''<br/>
 
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'''Friday'''<br/>
 
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'''Saturday'''<br/>
'''Saturday'''<br/>
 +
<div class="digestion">
 +
Digestion (Hashiya)<br/>
 +
・Sequencing analysis<br/>
 +
　We found sequence of chitinase A1 gene is correct.
 +
</div>
'''Sunday'''<br/>
'''Sunday'''<br/>
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Capture(Kusaba, Hara):the experiment about flies' phototaxis ②　♂:E.coli×4, ♀:E.coli×4
Capture(Kusaba, Hara):the experiment about flies' phototaxis ②　♂:E.coli×4, ♀:E.coli×4
</div>
</div>
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'''Tuesday'''<br/>
 
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'''Wednesday'''<br/>
 
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'''Thursday'''<br/>
 
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'''Friday'''<br/>
 
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'''Saturday'''<br/>
 
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'''Sunday'''<br/>
 

Latest revision as of 02:41, 6 October 2011

Contents

Lab Work

Week1: Monday 1st - Sunday 7th August

Week2: Monday 8th - Sunday 14th August

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.

Week3: Monday 15th - Sunday 21th August

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.

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

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

Friday

Capture(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

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

Sunday

Week5: Monday 29th August - Sunday 4th September

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-Gel0903.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-Gel0904.jpg
　We cultured rpoN-15 and Miniprep, but it did not contain rpoN gene.
・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
Kyoto-gel-09050.jpeg
・Screening PCR of rpoN
Kyoto-gel-09051.jpeg

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

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

Nutritional Signal(Hashiya)
・Screening PCR of glnL+G(4. sept)
Kyoto-gel-09060.jpeg
　We cultured glnL+G-9.
・Ligation
　We ligated rpoN to pSB1C3.


Wednesday

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

Nutritional Signal(Hashiya)
・Mini prep of glnL+G-9
　The concentration of DNA was 70.4ng/ul.
・Restriction of double terminator and glnL+G-9
　Cut double terminator with EcoRl and Xbal.
　Cut glnL+G-9 with EcoRl and Spel.
Kyoto-gel-09070.jpeg
　lane1:λ marker, lane2&3:glnL+G cut with E&S, lane4:double terminator cut with E&X, lane5&6:pSB1A3 cut with E&S

Thursday

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

Nutritional Signal(Hashiya)
・Screening PCR of rpoN
Kyoto-gel-09071.jpeg
　We cultured rpoN-9 and rpoN-11

Thursday

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

Nutritional Signal(Hashiya)
・Sequence
　We tryed to get sequence of sigma54 promoter-5, glnL-5, glnG-4, rpoN-9 and glnL+G-9. However, they were too thin to get sequence. So, we cultured all of them.


Friday

Nutritional Signal(Hashiya)
・Restriction
　Before getting sequence, we cut plasmid to know whether it is correct plasmid.
　We cut rpoN-9 with Hindlll, BamHl & Pstl.
　We also cut Lux operon with Hindll, EcoRl & Pstl.
Kyoto-gel09100.jpeg
　lane1 & 2:1kb DNA ladder and 100bp DNA ladder, lane3,4:digested rpoN-9, lane5,6,7&8:digested lux operon
　From this photo, we noticed lux operon were correct but rpoN-9 has some problem.


Saturday

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

Nutritional Signal(Hashiya)
・Mini prep
　The concentration of DNA were
　sigma 54 promoter 135.7 ng/ul
　glnL-5 143.2 ng/ul
　glnG-4 126.2 ng/ul
　glnL+G-9 25.7 ng/ul
　rpoN-10 102.0 ng/ul
　rpoN-11 95.1 ng/ul
・Restriction
　Before getting sequence, we cut plasmid to know whether it is correct plasmid.
　We cut rpoN-9, rpoN-10 and rpoN-11 with EcoRl & Pstl.
Kyoto-gel09110.jpeg
lane1&2:1kb DNA ladder and 100bp DNA ladder, lane5:digested rpoN-9, lane6:digested rpoN-10, lane7:digested rpoN-11
　This photo shows rpoN-9 has some problem.
・inverse PCR
　To erase restriction site of Pstl in rpoN, we did inverse PCR.
　Primer1:gcaagatgccaaatggttgatcaagagtc
　Promer2:agattgctgcggataaactgg

Sunday

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

Week7: Monday 12th September - Sunday 18th September

Monday

Capture: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

Nutritional Signal(Hashiya)
・Sequencing
　We found glnG-4 is correct. The others, sigma 54 promoter, glnL-5 and glnL+G-9 are not correct.

Tuesday

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

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Nutritional Signal(Hashiya)
・Screening PCR of glnL
Kyoto-gel0913.jpeg
　We cultured lane2's strain as glnL-9.

Wednesday

Nutritional Signal(Hashiya)
・Mini prep of glnL-9
　The concentration of DNA was 136.1 ng/ul
・Sequencing
　We found glnL-9 is correct.

Thursday

Nutritional Signal(Hashiya)
・PCR amplification of sigma 54 promoter
　We remake sigma 54 promoter.
　DNA oligo1:cggaattcgcggccgcttctagagattgca
　DNA oligo2:gaatgttgcaccaatatagtgcttcaatggaaacattaagcaccatgttggtgcaatctctagaagcggc
　DNA oligo3:gaatgttgcaccaatatagtgcttcaatggaaacattaagcaccatgttggtgcaatctctagaagcggc
　DNA oligo4:ggactagtaaaagcgaaatctgtgccaacttttaaattgcccctaaaaggcgttatcatgcgcacc
・Restriction of sigma 54 promoter.
　We cut PCR product with EcoRl and Spel.
・Gel extraction of sigma 54 promoter
Kyoto-gel0916.jpeg
　After gel extraction, the concentration of DNA was 18.7 ng/ul.
　The concentration of DNA was 136.1 ng/ul
・Screening PCR of rpoN
Kyoto-gel09161.jpeg
Kyoto-gel09162.jpeg

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.

Nutritional Signal(Hashiya)
・Screening PCR of rpoNm
　Kyoto-gel0917.jpeg
　Kyoto-gel09171.jpeg
　We cultured lane7's strain as rpoNm-11

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.

Nutritional Signal(Hashiya)
・Restriction of rpoNm-11
　We cut rpoNm-11 with EcoRl and Pstl.
　Kyoto-gel0918.jpeg
　This photo shows the restriction site of rpoN were relieved.

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

Tuesday

Digestion (Mori)

Transformation

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

Nutritional Signal(Hashiya)
・Restriction of glnL and glnG
　We cut glnL and glnG with EcoRl and Spel
・Gel extraction of glnL and glnG
Kyoto-gel-0920.jpeg
lane1:1kb DNA ladder, lane2&3:glnL, lane4&5:glnG
　After gel extraction, the concentration of DNA were
　glnL:18.7 ng/ul
　glnG:10.0 ng/ul
・Sequencing of rpoNm-11
　We found rpoNm-11 is correct.

Wednesday

Digestion (Mori, Nobeyama)

Colony PCR Performed colony PCR to check if ligation and transformation were successful. Gel electrophoresis shown that the ligation and transformation of pSB1C3 with ChiA was successful.

Saturday

Digestion (Hashiya)
・Sequencing analysis
　We found sequence of chitinase A1 gene is correct.

Sunday

Capture(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

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

Retrieved from "http://2011.igem.org/Team:Kyoto/Lab_Work"