Team:TMU-Tokyo/Notebook/Assay 3 Protocol and Result

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<Br>
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<B>■Assay</B><Br>
<B>■Assay</B><Br>
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Device1 Assay<Br>
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<A Href="https://2012.igem.org/Team:TMU-Tokyo/Notebook/Assay_0_Protocol_and_Result">Assay0</A><Br>
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Device2 Assay<Br>
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<A Href="https://2012.igem.org/Team:TMU-Tokyo/Notebook/Assay_1_Protocol_and_Result">Device1 Assay</A><Br>
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<A Href="https://2012.igem.org/Team:TMU-Tokyo/Notebook/Assay_2_Protocol_and_Result">Device2 Assay</A><Br>
Device3 Assay<Br>
Device3 Assay<Br>
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<p class="description">
<p class="description">
<b>■Assay3 Protocol</b><Br>
<b>■Assay3 Protocol</b><Br>
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<Br>1The cells were cultured at 30 ℃ for 18 hours in medium<Br>
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<Br>Ⅰ. Extraction of the crude enzyme solution<Br>
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2Centrifuged 10000 × g　5min<Br>
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<Br>
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3Remove the supernatant<Br>
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<p class="description3">
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4Wash the fungus<Br>
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<b>1. </b>The cells were cultured at 30 ℃ for 18 hours in medium<Br>
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E. coli disrupted by sonicator in buffer of 50mM Tris-HCl pH7.2 .<Br>
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<b>2. </b>Centrifuged 10000 × g　5min<Br>
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Centrifuged 10000 × g at 4 ℃ so that the enzyme is not deactivated, the supernatant was harvested as crude enzyme solution (If you want to save is stored at 4 ℃ in a cold room)
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<b>3. </b>Remove the supernatant<Br>
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<b>4. </b>Wash the fungus<Br>
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      Add dw and Centrifuged 10000 × g　5min<Br>
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<b>5. </b>Remove the supernatant<Br>
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<b>6. </b>E. coli disrupted by sonicator in buffer of 50mM Tris-HCl pH7.2 .<Br>
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<b>7. </b>Centrifuged 11000 × g at 4 ℃ so that the enzyme is not deactivated, the supernatant was harvested as crude enzyme solution (If you want to save is stored at 4 ℃ in a cold room)<Br>
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<Br>
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The reaction mixture are
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50mM Tris-HCl ph7.2 0.8ml<Br>
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There sodium formate 20mM 0.1ml (2mM final concentration)<Br>
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20mM NAD + 0.1ml<Br>
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Incubated for 5 minutes at 37 ℃ put<Br>
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I was allowed to react at 37 ℃ in the next, put 0.05ml crude enzyme solution.<Br>
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Formic acid formic acid was quantified through the reaction mixture liquid chromatography.<Br>
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<Br>
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<Br>
</p>
</p>
<p class="description">
<p class="description">
<b>■Assay3 Result</b><Br>
<b>■Assay3 Result</b><Br>
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<Br>
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<p class="description3">
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only formate 2mM<Br>
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Peak = 8.81(min)<Br>
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<Br>
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<BR>
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<b>BBa_K749015</b>
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<Br>
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<p class="description">
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<table border>
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<tr>
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<td><p class="description"> Reaction time </td>
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<td> volume </td>
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<td> Peak area </td>
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<td> Peak area(%) </td>
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</tr>
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<tr>
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<td>30min </td>
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<td>50μl</td>
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<td>229371.2</td>
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<td>80.88</td>
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</tr>
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<tr>
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<td>10min</td>
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<td>20μl</td>
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<td>248294.2</td>
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<td>83.9823</td>
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</tr>
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</table>
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<Br>
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<p class="description3">
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<b>WT</b><Br>
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<table border>
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<tr>
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<td><p class="description"> Reaction time </td>
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<td> volume </td>
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<td> Peak area </td>
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<td> Peak area(%) </td>
 +
</tr>
 +
<tr>
 +
<td>10min </td>
 +
<td>20μl</td>
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<td>232791.6</td>
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<td>83.691</td>
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</tr>
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</table>　
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<Br>
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<p class="description3">
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<b>No enzym</b><Br>
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<table border>
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<tr>
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<td><p class="description"> Reaction time </td>
 +
<td> volume </td>
 +
<td> Peak area </td>
 +
<td> Peak area(%) </td>
 +
</tr>
 +
<tr>
 +
<td>10min </td>
 +
<td>20μl</td>
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<td>243418.6</td>
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<td>84.37</td>
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</tr>
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</table>　
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          <Br>
</p>
</p>
<Br>
<Br>

Latest revision as of 02:24, 27 September 2012

 




■Experiment
1st week (8.13 - 8.19)
2nd week (8.20 - 8.26)
3rd week (8.27 - 9. 2)
4th week (9. 3 - 9. 9)
5th week (9.10 - 9.16)
6th week (9.17 - 9.23)
7th week (9.24 - 9.30)


■Protocols


■Assay
Assay0
Device1 Assay
Device2 Assay
Device3 Assay




Assay 3


■Assay3 Protocol

Ⅰ. Extraction of the crude enzyme solution

1. The cells were cultured at 30 ℃ for 18 hours in medium
2. Centrifuged 10000 × g　5min
3. Remove the supernatant
4. Wash the fungus
Add dw and Centrifuged 10000 × g　5min
5. Remove the supernatant
6. E. coli disrupted by sonicator in buffer of 50mM Tris-HCl pH7.2 .
7. Centrifuged 11000 × g at 4 ℃ so that the enzyme is not deactivated, the supernatant was harvested as crude enzyme solution (If you want to save is stored at 4 ℃ in a cold room)

The reaction mixture are 50mM Tris-HCl ph7.2 0.8ml
There sodium formate 20mM 0.1ml (2mM final concentration)
20mM NAD + 0.1ml
Incubated for 5 minutes at 37 ℃ put
I was allowed to react at 37 ℃ in the next, put 0.05ml crude enzyme solution.
Formic acid formic acid was quantified through the reaction mixture liquid chromatography.


■Assay3 Result

only formate 2mM
Peak = 8.81(min)


BBa_K749015

Reaction time

volume Peak area Peak area(%)
30min 50μl 229371.2 80.88
10min 20μl 248294.2 83.9823

WT

Reaction time

volume Peak area Peak area(%)
10min 20μl 232791.6 83.691
　

No enzym

Reaction time

volume Peak area Peak area(%)
10min 20μl 243418.6 84.37
　



 

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