Part:BBa_K3150000
AzoR
We optimized the codons of AzoR(BBa_K1336000,uploaded by iGEM14_UCL ) to match RFC10. AzoR is a azoreductase derived from Pseudomonas putida.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 340
Illegal NgoMIV site found at 367 - 1000COMPATIBLE WITH RFC[1000]
SDS-page Verification
SDS-PAGE experiments were performed to verify the expression of the enzyme protein. AzoR-, AzoR+ represent no induction and after induction by IPTG. By reviewing the literature, we found that AzoR has a protein size 23.1 kDa. After comparison with the images, we found that all proteins were successfully expressed under the induction of IPTG.
Decolorization Ability Test
We tested the decolorization ability of AzoR. However, the decolorization function needs to be carried out in the oxygen free condition, and the experimental conditions are strict, so the experiment did not go well. In order to simulate the anaerobic environment, we put each group of bacterial solution into EP pipe and sealed it with sealing strip. The supernatant was detected by absorbance every certain time. However, no matter whether there is IPTG induction or not, we have not seen obvious degradation of dyes. Then we looked up the literature and found that CuCl2 may work normally only when it exists. So we did two more experiments, Azor + added CuCl2, AzoR - did not add CuCl2. After two days of experiment, we got disappointed data again. After a simple statistic of this set of data, we see two very gentle curves (Figure 1). No matter whether CuCl2 is added or not, the absorbance of the dye decreases slightly, which is consistent with the degradation of the dye itself, but the decrease of CuCl2 is slightly larger, which indicates that the presence of CuCl2 can promote the catalytic effect of AzoR to some extent, but after 48 hours, the absorbance of the supernatant of the two groups still hovers around 1.5, which indicates that Azor is not in our experimental condition. Show excellent decolorization ability.
| None |