Difference between revisions of "Part:BBa K2275007"
 
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<partinfo>BBa_K2275007 short</partinfo>
 
<partinfo>BBa_K2275007 short</partinfo>
  
 
This composite part is composed of R0010, B0034 and K2275001 which encodes nitrite reductase derived from E. coli K-12 MG1655 strain.  
 
This composite part is composed of R0010, B0034 and K2275001 which encodes nitrite reductase derived from E. coli K-12 MG1655 strain.  
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Then we used Griess reagent assay to test the activity of NiR. The principle and mechanism of Griess reaction are shown below.  
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Then we used Griess reagent assay to test the activity of NiR/MG1655. The principle and mechanism of Griess reaction are shown below.  
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[[File:Griess reagent assay mechanism.png|350px||centre]]
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[[File:Griess reagent assay mechanism.png|350px|centre]]
  
 
When the broth contains nitrite, by adding the Griess reagent which consists of naphthylethylenediamine dihydrochloride, sulphanilamide,  phosphoric acid can form a diazonium salt with nitrite. The diazonium salt will have absorbance at wavelength 545 nm. By the reaction, we can further calculate the nitrate conversion.
 
When the broth contains nitrite, by adding the Griess reagent which consists of naphthylethylenediamine dihydrochloride, sulphanilamide,  phosphoric acid can form a diazonium salt with nitrite. The diazonium salt will have absorbance at wavelength 545 nm. By the reaction, we can further calculate the nitrate conversion.
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In view of concerning with self-degradation of nitrite, we also do the blank experiment. The result is shown below. The bar in light green indicates the nitrite concentration initially, and the bar in blue indicated concentration after 4 hours incubation.
 
In view of concerning with self-degradation of nitrite, we also do the blank experiment. The result is shown below. The bar in light green indicates the nitrite concentration initially, and the bar in blue indicated concentration after 4 hours incubation.
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[[File:Griess calibration curve.png|500px||centre]]
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[[File:Griess calibration curve.png|500px|centre]]
 
From blank experiment, the result indicated that nitrite concentration maintained the same before and after 4 hour. Figure below shows the conversion of nitrite that catalyzed by NiR is pretty high. The result approve that nir gene has the ability to convert nitrite.  
 
From blank experiment, the result indicated that nitrite concentration maintained the same before and after 4 hour. Figure below shows the conversion of nitrite that catalyzed by NiR is pretty high. The result approve that nir gene has the ability to convert nitrite.  
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[[File:Conversion_of_NiR.png|450px||centre]]
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[[File:Conversion of NiR.png|450px|centre]]
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Latest revision as of 07:27, 28 October 2017

PlacI-B0034-NiR cluster

This composite part is composed of R0010, B0034 and K2275001 which encodes nitrite reductase derived from E. coli K-12 MG1655 strain. Then we used Griess reagent assay to test the activity of NiR/MG1655. The principle and mechanism of Griess reaction are shown below.

Griess reagent assay mechanism.png

When the broth contains nitrite, by adding the Griess reagent which consists of naphthylethylenediamine dihydrochloride, sulphanilamide, phosphoric acid can form a diazonium salt with nitrite. The diazonium salt will have absorbance at wavelength 545 nm. By the reaction, we can further calculate the nitrate conversion. In our experiment, we use 1-naphthylamine instead of naphthylethylenediamine dihydrochloride. Before starting our test, we need to do the calibration curve between the absorbance at wavelength 545 nm and different nitrite concentration. The result was shown below. According to the calibration curve, we can deduce the nitrite concentration from absorbance in different time.

In view of concerning with self-degradation of nitrite, we also do the blank experiment. The result is shown below. The bar in light green indicates the nitrite concentration initially, and the bar in blue indicated concentration after 4 hours incubation.

Griess calibration curve.png

From blank experiment, the result indicated that nitrite concentration maintained the same before and after 4 hour. Figure below shows the conversion of nitrite that catalyzed by NiR is pretty high. The result approve that nir gene has the ability to convert nitrite.

Conversion of NiR.png

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 286
    Illegal BglII site found at 1287
    Illegal BglII site found at 2070
    Illegal BamHI site found at 567
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2093
    Illegal AgeI site found at 551
    Illegal AgeI site found at 947
    Illegal AgeI site found at 2105
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 401