Difference between revisions of "Part:BBa K3599009"
 
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===Sequence and Features===
 
===Sequence and Features===
 
<partinfo>BBa_K3599009 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3599009 SequenceAndFeatures</partinfo>
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===Design & Experience===
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====SDS-PAGE Verification of Nitrilase====
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We performed SDS-PAGE verification on the fermentation broth of the engineered strain after induction, and the results are shown below.<br>
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[[File:T--TPR China--SDS.png|600px|thumb|center|The SDS-PAGE result of fermentation broth]]<br>
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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.
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====Nitrilase Enzyme Activity====
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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 NH<sub>4</sub><sup>+</sup> produced by PAN degradation.<br>
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<b>I. Standard Curve</b><br>
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We plotted the standard curve of the ammonium concentration. The experimental sample's NH<sub>4</sub><sup>+</sup> concentration can thus be calculated from the standard concentration equation.<br>
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[[File:T--TPR China--StandardCurve.png|600px|thumb|center|Standard curve of NH<sub>4</sub><sup>+</sup>]]<br>
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<b>II. Nitrilase Enzyme Activity</b><br>
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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.<br>
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[[File:T--TPR China--EnzymaticPhoto.png|600px|thumb|center|Experimental phenomena of enzyme activity test]]<br>
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[[File:T--TPR China--EnzymaticData.png|600px|thumb|center|Enzymatic activity of nitrilase]]<br>
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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.

Latest revision as of 06:35, 27 October 2020


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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 722
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE 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.

The SDS-PAGE result of fermentation broth

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.

Standard curve of NH4+

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.

Experimental phenomena of enzyme activity test

Enzymatic activity of nitrilase

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.