Difference between revisions of "Part:BBa K3599003"
 
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===Design & Experience===
 
===Design & Experience===
 
In iGEM2020 TPR_China Project, the adNIT was expressed using pTac system, which was pre-embedded into commercial expression vector pET-28b. The whole expression cassette can be found in [[Part:BBa_K3599009|BBa_K3599009]].
 
In iGEM2020 TPR_China Project, the adNIT was expressed using pTac system, which was pre-embedded into commercial expression vector pET-28b. The whole expression cassette can be found in [[Part:BBa_K3599009|BBa_K3599009]].
 +
 +
====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.<br>
 +
[[File:T--TPR China--SDS.png|600px|thumb|center|The SDS-PAGE result of fermentation broth]]<br>
 +
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 NH<sub>4</sub><sup>+</sup> produced by PAN degradation.<br>
 +
<b>I. Standard Curve</b><br>
 +
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>
 +
[[File:T--TPR China--StandardCurve.png|600px|thumb|center|Standard curve of NH<sub>4</sub><sup>+</sup>]]<br>
 +
<b>II. Nitrilase Enzyme Activity</b><br>
 +
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>
 +
[[File:T--TPR China--EnzymaticPhoto.png|600px|thumb|center|Experimental phenomena of enzyme activity test]]<br>
 +
[[File:T--TPR China--EnzymaticData.png|600px|thumb|center|Enzymatic activity of nitrilase]]<br>
 +
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.
  
  
 
===Reference===
 
===Reference===
 
Novikov, A. D. , Riabchenko, L. E. , Leonova, T. E. , Larikova, G. A. , & Yanenko, A. S. . (2016). A bacterial strain alcaligenes denitrificans С-32 contains two nitrilases with different substrate specificities. [https://www.researchgate.net/publication/316711055_A_Bacterial_Strain_Alcaligenes_denitrificans_S-32_Contains_Two_Nitrilases_with_Different_Substrate_Specificities 10.21519/0234-2758-2016-32-6-45-52]
 
Novikov, A. D. , Riabchenko, L. E. , Leonova, T. E. , Larikova, G. A. , & Yanenko, A. S. . (2016). A bacterial strain alcaligenes denitrificans С-32 contains two nitrilases with different substrate specificities. [https://www.researchgate.net/publication/316711055_A_Bacterial_Strain_Alcaligenes_denitrificans_S-32_Contains_Two_Nitrilases_with_Different_Substrate_Specificities 10.21519/0234-2758-2016-32-6-45-52]

Latest revision as of 06:05, 27 October 2020


adNIT


Overview

Nitrilase coming from Achromobacter denitrificans, catalysis of the reaction: nitrile + 2H2O = carboxylate + NH3. The codon usage was optimized for expression in Escherichia coli.


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 629
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 191
    Illegal BsaI site found at 374


Primitive Species Background

The type species of gram negative, aerobic bacteria in the genus. A genus of strictly aerobic, non-spore forming rods. Soil and water are regarded as the natural habitat. They are sometimes isolated from a hospital environment and humans. Previously in the genus Alcaligenes, the classification and nomenclature of this species has been frequently emended. The two subspecies, Achromobacter xylosoxidans subsp. denitrificans and Achromobacter xylosoxidans subsp. xylosoxidans are associated with infections.


Design & Experience

In iGEM2020 TPR_China Project, the adNIT was expressed using pTac system, which was pre-embedded into commercial expression vector pET-28b. The whole expression cassette can be found in BBa_K3599009.

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.


Reference

Novikov, A. D. , Riabchenko, L. E. , Leonova, T. E. , Larikova, G. A. , & Yanenko, A. S. . (2016). A bacterial strain alcaligenes denitrificans С-32 contains two nitrilases with different substrate specificities. 10.21519/0234-2758-2016-32-6-45-52