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Team:TMU-Tokyo/Notebook/Assay 3 Protocol and Result
From 2012.igem.org
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<p class="description"> | <p class="description"> | ||
<b>■Assay3 Protocol</b><Br> | <b>■Assay3 Protocol</b><Br> | ||
| - | <Br>Ⅰ.Extraction of the crude enzyme solution<Br> | + | <Br>Ⅰ. Extraction of the crude enzyme solution<Br> |
<Br> | <Br> | ||
| - | <b>1.</b>The cells were cultured at 30 ℃ for 18 hours in medium<Br> | + | <p class="description3"> |
| - | <b>2.</b>Centrifuged 10000 × g 5min<Br> | + | <b>1. </b>The cells were cultured at 30 ℃ for 18 hours in medium<Br> |
| - | <b>3.</b>Remove the supernatant<Br> | + | <b>2. </b>Centrifuged 10000 × g 5min<Br> |
| - | <b>4.</b>Wash the fungus<Br> | + | <b>3. </b>Remove the supernatant<Br> |
| + | <b>4. </b>Wash the fungus<Br> | ||
Add dw and Centrifuged 10000 × g 5min<Br> | Add dw and Centrifuged 10000 × g 5min<Br> | ||
| - | <b>5.</b>Remove the supernatant<Br> | + | <b>5. </b>Remove the supernatant<Br> |
| - | <b>6.</b>E. coli disrupted by sonicator in buffer of 50mM Tris-HCl pH7.2 .<Br> | + | <b>6. </b>E. coli disrupted by sonicator in buffer of 50mM Tris-HCl pH7.2 .<Br> |
| - | <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> | + | <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> |
<Br> | <Br> | ||
The reaction mixture are | The reaction mixture are | ||
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I was allowed to react at 37 ℃ in the next, put 0.05ml crude enzyme solution.<Br> | I was allowed to react at 37 ℃ in the next, put 0.05ml crude enzyme solution.<Br> | ||
Formic acid formic acid was quantified through the reaction mixture liquid chromatography.<Br> | Formic acid formic acid was quantified through the reaction mixture liquid chromatography.<Br> | ||
| - | + | <Br> | |
| + | <Br> | ||
</p> | </p> | ||
<p class="description"> | <p class="description"> | ||
<b>■Assay3 Result</b><Br> | <b>■Assay3 Result</b><Br> | ||
<Br> | <Br> | ||
| + | <p class="description3"> | ||
only formate 2mM<Br> | only formate 2mM<Br> | ||
Peak = 8.81(min)<Br> | Peak = 8.81(min)<Br> | ||
| - | + | <Br> | |
| + | <BR> | ||
<b>BBa_K749015</b> | <b>BBa_K749015</b> | ||
<Br> | <Br> | ||
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<Br> | <Br> | ||
<Br> | <Br> | ||
| + | <p class="description3"> | ||
<b>WT</b><Br> | <b>WT</b><Br> | ||
<table border> | <table border> | ||
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</table> | </table> | ||
<Br> | <Br> | ||
| + | <p class="description3"> | ||
<b>No enzym</b><Br> | <b>No enzym</b><Br> | ||
<Br> | <Br> | ||
Revision as of 02:05, 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
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 |
