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

 

■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

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■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