Atrazine is one of the most commonly used herbicides in the United States: Approximately 80 million pounds of it is used across the country each year.Nevertheless, there is a serious environmental problem.
Agricultural pollution is the pollution of the environment and related surroundings caused by using natural and chemical products in agriculture. The pollution sources are harmful to all the living organisms that obtain energy from the food cultivated on farms. Agricultural pollution tends to be more serious since the demand for food is increasing, proportional to the increase in population.
Atrazine, which is produced by an agrochemical company named Syngenta in Swiss, was first registered for use as an agricultural herbicide in the United States in 1959. Besides being used by farmers to control broadleaf weeds and grasses that interfere with the growth of crops, it is also used as a weed killer on golf courses as well as a variety of commercial and residential lawns. It is now the second most used herbicide in the US after glyphosate, since approximately 80 million pounds of this herbicide is used across the country each year. The large amount usage of Atrazine making it common pollution of both soil, drinking water, people, and wildlife. So, it has been banned to use in the European Union.
It has been reported that Atrazine can induce significant health problems including increased risk of prostate cancer, decreased sperm count in men, and a higher risk of breast cancer in women. It is an endocrine disruptor that directly affects the sexual development of amphibians by changing their hormone cycle.
Therefore, using Atrazine too much can also lead to declines of endangered amphibians and many other endangered species throughout the country.
However, owing mainly to the superior weeding efficacy and low price of Atrazine, the current policy in most countries is reinforcing management instead of explicitly restricting the use of it. Methods we currently used for detecting the amount of Atrazine are Gas chromatography-mass spectrometry (GC-MS) and Liquid chromatography-mass spectrometry (LC-MS). These detecting methods have evident limitations such as high cost, not convenient to carry, and hard to operate. Therefore, our goals are to develop a biosensor for Atrazine detection using the theory of synthetic biology. It must be simple and convenient, and at the same time high sensitivity towards Atrazine.
The entire experiment went well. AtzR, PatzD, GFP, and T7 Terminator genes were cloned successfully. We then extracted the pME6032 and pBBR1MCS-2 plasmid and measured the concentration of them using Nanodrop. pME6032 plasmid and AtzR genes were digested with restriction enzymes KpnI and XhoI. PBBR1MCS-2 plasmid and PatzD-GFP-T7 terminator genes were digested with restriction enzymes EcoRI and BamHI. We used the gel recovery kit to purify and recycle the enzyme digestion products. Next, we used the T4 DNA ligase to construct the two vectors. At the same time, the pBBR1MCS-2 plasmid was linearized using PCR and the product was recycled by gel recovery kit (the linearized vector could be constructed by homologous recombination).
The constructed vectors were transformed into E. coli competent cell DH5 by heat shock and the obtained monoclonals were verified using PCR. The monoclonals with specific bands were sequenced and the vector with the correct sequence was mixed with 50% glycerol by volume and placed at -80℃ for preservation.
Pseudomonas putida competent cells were prepared, then plasmid pME6032-AtzR and pBBR1MCS-2-PatZD-GFP-T7 terminator were transformed by electroshock.
At the last stage, different concentrations of cyanuric acid were added into the functional testing system for functional testing. The GFP fluorescence value and bacterial OD600 value were collected by a microplate reader, and finally, the experimental results were analyzed and plotted.
Theoretically, when there is Atrazine detected, the reporter gene will express a GFP protein that can be detected by naked eyes under the condition of excitation spectrum, meaning that we construct the biosensor we want successfully.
We hope that in the future, our product will help millions of people to know whether their farmlands or drinking water are Atrazine polluted.
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