Considering the limitations of laboratory conditions, some data cannot be well measured and recorded (such as OD value and fluorescent photo taking), so the data are sorted out and the repeatable and meaningful data are selected for analysis as follows.
Determination of the detectable range of phosphate concentration of PhoA-GFP Escherichia coli.
In this experiment, three initial concentrations of 0mM, 0.2mM, and 1mM were set to preliminarily predict the detection range of PhoA-GFP E. coli phosphate concentration. The experimental results showed that different concentrations of phosphate were added to the culture medium for treatment, within 15 minutes, compared with the control group, the E. coli containing PhoA-GFP plasmid had a higher detectable fluorescence value in the 0-0.2mM concentration range. Big difference. This result not only verifies that PhoA protein expression in E. coli is indeed induced by phosphorus starvation, but also provides a basis for us to further set the PhoA-GFP phosphate detection range in more detail.
(2) Determination of phosphate detection time for PhoA GFP Escherichia coli.
According to the results of previous tests. We found that the detectable fluorescence values of PhoA-GFP were significantly different in the concentration range of 0-0.2mM. In other words, in the case of phosphorus deficiency, the detectable fluorescence of PhoA GFP E. coli was the most significant difference compared with the control group. Therefore, we in this condition, the Escherichia coli containing PhoA -GFP plasmid and contrasting ck contains only GFP Escherichia coli in culture, then sampling at different time points, take time to deal with different PhoA-GFP Escherichia coli compared with ck GFP fluorescence detection of e. coli, the results showed that with the passage of time, the GFP fluorescence signal intensity difference decreases. This suggests that PhoA-GFP E. coli can detect phosphorus deficiency signals in a short period of time, thus providing the possibility for us to use it to rapidly detect the presence of phosphorus pollution in water.
Results & Conclusions:
Based on these experimental results, it can be concluded that (1) PhoA gene is indeed affected by phosphate, and the PhoA gene expression level decreases with the increase of phosphate concentration in the water environment. (2) At the same time, PhoA gene expression would be stimulated and inhibited at the very beginning if it was in a water environment polluted by phosphate.
Through this experiment, our experimental results have reached preliminary expectations. However, there are still many areas for improvement in this experiment. We will continue to carry out the project in the future. We will continue to improve the experimental methods and conditions. We can modify the existing governance vectors and find corresponding solutions to improve the biological detection. Sensitivity and accuracy, such as repairing and improving the available PhoA sequence, observing the protein structure to find its key domains, using more effective reporter genes, or modifying reporter genes such as GFP to increase the response intensity of fluorescent signals, etc. Wait.
In the future, we will be able to make the biological sensor PhoA-GFP more accurate, so that the biological phosphorus sensing system can not only monitor whether the water environment is polluted by phosphorus nutrition, but also detect the degree of phosphorus pollution in the water body and the length of the pollution time. , It can even be made into test strips. On the one hand, it is conducive to storage and carrying, on the other hand, it can make environmental monitoring personnel more convenient and quickly to detect... With such a phosphorus sensor, people can understand their living environment Whether the water resources of China are polluted by phosphorus or not, and then help people pay attention to environmental protection, to actively protect water resources and care for the environment.
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