Part:BBa_K2817007
PyeaR-RBS-amilCP
The promoter PyeaR is sensitive to nitrate and nitrite. When nitrate and nitrite enter E. coli, they are converted to nitric oxide. Nitric oxide binds to the repressor protein NsrR, which inactivates PyeaR to inhibit transcription of downstream genes. Then the promoter PyeaR will be activated to express the amilCP.
1. Usage and Biology
We learned that iGEM 2010 Team BCCS-Bristol used BBa_K381001 to detect nitrate and nitrite in the soil. The content of nitrate and nitrite in the soil can reflect the fertility of the soil. Farmers can determine which soils are fertile by detecting the fluorescence of GFP. In this way, farmers only need to apply fertilizer in places where there is no fertility, which can save excess fertilizer. Given the economic costs and the impact of eutrophication on ecosystems, the use of BBa_K381001 has great benefits for both farmers and the environment. However, due to the influence of outdoor temperature, GFP fluorescence fluctuates greatly. This instability is unfavorable for the detection of soil fertility. In addition, the detection of GFP fluorescence requires special equipment that is not readily available to farmers. Therefore, we replaced GFP with blue chromoprotein amilCP for visual detection of soil fertility. On the one hand, amilCP expression is less affected by temperature and is a more stable reporter than GFP. On the other hand, amilCP does not require special equipment to be visible to the naked eye. Therefore, we believe that our improved part BBa_K2817007 is very beneficial to farmers.
2. Characterization
According to the results of the ShanghaiTechChina_B 2016 team, 100μM SNP aqueous solution can continuously release NO, and the NO concentration is stable at about 5.5μM. Since our project also tested for inflammatory signals, we chose this concentration before testing for BBa_K381001 and BBa_K2817007.
The construction of BBa_K381001 can be seen from Figure 1A. We transformed the plasmid containing BBa_K381001 into DH5α, and cultured at 37 ℃ overnight to dilute to OD = 0.4. Then we took half as control and the other half added SNP aqueous solution and induced at 37 ℃ for 6 h. Then we detected the fluorescence using a microplate reader and a fluorescence microscope (Figure 1B, 1C). We can see that PyeaR can be effectively activated by NO with almost no leakage.
Figure 1. The test of BBa_K381001. A, the construction of BBa_K381001. B, Histogram of GFP fluorescence: LB control, without SNP, with 100μM SNP. C, GFP fluorescence image from top to bottom: without SNP, with 100μM SNP.
The construction of BBa_K2817007 can be seen from Figure 2A. We transformed the plasmid containing BBa_K2817007 into DH5α, and cultured at 37 ℃ overnight to dilute to OD = 0.4. Then we took half as control and the other half added SNP aqueous solution and induced at 37 ℃ for 6 h. We also set up negative control group which doesn’t contain amilCP. After 6 h at 37 ℃, 1 mL of the bacterial solution was centrifuged at 8000 rpm for 1 min (Figure 2B). We can see the result of PyeaR being activated by NO by the naked eye.
Figure 2. The test of BBa_K2817007. A, the construction of BBa_K2817007. B, Pellets of bacteria transformed with plasmid containing BBa_K2817007 after induction of 6h. From left to right: negative control group, without SNP group, with 100μM SNP group.
3. Conclusion
In conclusion, we confirmed our improvement through an experimental comparison between the two parts. In the real world, our improved part BBa_K2817007 has better usability. In the future, we will further confirm the situation of different concentrations of NO and different temperature conditions.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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