Team:Kyoto/Lab Work

From 2011.igem.org

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