Team:ECNUAS/Proof Of Concept
Proof of Concept
Overview
Atrazine is the most widely used herbicide in the world today. In the middle of the
last century, it was developed and put into production. The excellent herbicidal efficacy and low price of
atrazine make it widely popularized.
Researches have shown that atrazine pollution has a gender reversal effect on
frogs, and it also has a negative effect on some other organisms that interfere with growth and
reproduction.
Inhalation of a small amount of atrazine can cause physical discomfort, such as
nausea and dizziness; if you stay in an environment contaminated by atrazine for a long time, the function
of your immune system and lymphatic system is very likely to be impaired. High-dose atrazine may cause
cancer.
To summarize, atrazine is toxic and easily enters the human body through food. It
is dangerous to the human nervous system, immune system, and reproductive system. Thus, it is also currently
listed as an international environmental priority control pollutant.
We position our final product as a quick and convenient detection device of
atrazine.
It is a cell-free atrazine biosensor enabling users to detect cyanuric acid, the metabolite of the herbicide
Atrazine, fast, easily, conveniently at an affordable price.
To realize our idea, we came up with the following design-creation of genetically
encoded biosensor to detect the concentration of CYA, which is a metabolite of the herbicide Atrazine. In
this biosensor system, protein atzR is first expressed, which has been previously reported as a CYA binding
protein and can regulate the promoter Pprovoin5. In the presence of CYA, atzR changes the binding
conformation with promoter Pprovoin5 upon binding with CYA, so as to activate the expression of downstream
reporter gene green fluorescent protein(GFP). The fluorescence signal of GFP can be detected by the
fluorescence detector and the concentration of CYA can be determined by signal intensity. In this system,
GFP can be further replaced by other visible chromogenic proteins, so as to realize the rapid visual
detection of CYA.
Supporting Experiment Results
We have successfully obtained an engineered bacteria (bacteria C) that contains the
two plasmids above. This engineered bacteria is used to make our preliminary functional test to verify the
feasibility of our biosensor idea.
Functional Tests
Table 1. Fluorescence intensity when the concentration of CYA equals to 30uM and the duration is 4
hours
Figure 1. Histogram of the fluorescence intensity when the concentration of CYA equals to 30uM and the
duration is 4 hours
Clearly as figure 1 indicated, compared with the blank control, bacteria C presents
obvious higher fluorescence reaction to the cyanuric acid, the metabolite of the herbicide. In such case, it
did work in detecting the cyanuric acid.
This experiment result is encouraging since it qualitatively proved the power of
our engineered bacteria in detecting cyanuric acid. This consolidates the foundation for producing our
future biosensor.
In addition, we also want to explore the concentration of cyanuric acid and the
detection effectiveness of our engineered bacteria.
Table 2. Fluorescence intensity of bacteria C when when the duration is 4 hours under different
concentration of the cyanuric acid
Figure 2. Histogram of the fluorescence intensity of bacteria C when the duration is 4 hours under
different concentration of the cyanuric acid
Figure 3. Histogram of the fluorescence intensity of bacteria C when the duration is 6 hours under
different concentration of the cyanuric acid
According to the histograms (Fig. 2 and Fig. 3), the fluorescence intensity shows a
decreasing trend with the increase of concentration of cyanuric acid when we used the bacteria C for tests.
Therefore, we infer that the cyanuric acid might affect the growth of strains so that the higher the
concentration of the cyanuric acid, the worse the growth of the bacteria, the less of the amount of the
effective “biosensor”. This experiment result provide us with some clues for optimizing our experiments.
Thus, in order to fully eliminate this impact, we consider about continuing the concept of cell-free
extraction and cell-free expression in the next stage of our project. As long as we obtain enough products
of cell-free extraction, we could step into the cell-free expression experiments to ensure the performance
of our biosensor without bacteria.
In the long run, we hope that we will use 3D printing to build the device which
could load our cell-free biosensor and conduct more function tests with it. In the meantime, as the buffer
in cell-free expression needs to be freshly configured, it is necessary to explore ways to preserve the
product if it is made into a portable detection device.
Finally, we hope to carry our device to detect the content of cyanuric acid in
lake/river water and other environmental water.