Part:BBa_K2275007
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.
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.
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.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE 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 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE 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 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 401
| None |