Part:BBa_K3599009
pTac-adNIT
The expression cassette of adNIT (Part:BBa_K3599003), constructed on the commercial expression vector pET-28b, which uses pTac promoter to control the expression level of protein of interest.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 722
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI site found at 284
Illegal BsaI site found at 467
Design & Experience
SDS-PAGE Verification of Nitrilase
We performed SDS-PAGE verification on the fermentation broth of the engineered strain after induction, and the results are shown below.
Electrophoresis results show that our sample has a specific band around 31KD compared to the negative control, this proves that the engineered bacteria we constructed have expressed nitrilase protein.
Nitrilase Enzyme Activity
The nitrilase has PAN as substrate to degrade PAN into phenylacetic acid and ammonium. Ammonium and Nessler's Reagent can produce reddish-brown precipitation. The shade is proportional to the concentration of NH4+ produced by PAN degradation.
I. Standard Curve
We plotted the standard curve of the ammonium concentration. The experimental sample's NH4+ concentration can thus be calculated from the standard concentration equation.
II. Nitrilase Enzyme Activity
The activity of nitrilase was verified by detecting the ammonium ions produced by the degradation of PAN by the bacterial liquid. We define U as the amount of PAN that can be degraded per microliter per hour.
As can be seen, the U value of pTac-laNIT is 0.6115, which is 1.27 times that of the negative control;the U value of pTac-adNIT is 0.6205, which is 1.3 times that of the negative control.These data indicate that our engineered bacteria have high activity of nitrilase.
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