Team:ECNUAS/Description

ECNUAS

Project Description

I. Background

With the development of science and technology, agriculture becomes the most crucial domain. It plays a vast role in the national construction and becomes the basis impetus to develop the whole society. Agriculture is not only an indicator of the strength of a country, but also shows the living conditions of the people.

Agricultural chemicals are the indispensable part of the development of agriculture. But there are still many problems about the agricultural chemicals. So this year, our title is to test the content of cyanuric acid in atrazine.

Atrazine is one of the most widely used herbicides in the world. It is cheap and also effective on many crops. But it still has negative effects. Atrazine will leave a residue in the soil called cyanuric acid, it has endocrine disruption, genotoxicity, reproductive toxicity and other harm to mammals. Figure 1 shows the process of cyanuric acid accumulation. So we are going to design portable instrument to detect the content of residue in herbicide. lt should be cheap and convenient.

Figure 1. The process of Atrazine biodegradation.

II. General concept

On the principle of this sensor. By combining cyanuric acid with a specific plasmid, it activates the promoter in the body, which activates the downstream gene ,and induces gene expression to show the signs of fluorescence. The content of cyanuric acid is detected by detecting the fluorescence concentration. Our general concept is shown in Figure 2.

Figure 2. Genetic design of our biosensor

So what we have to do is to combine the reporter gene amil_GFP and pUC57_mini_Pprovoin5 to obtain plasmid B. After that, the two plasmids, plasmid B (pUC57_mini_Pprovoin5_amilGFP), and plasmid A (pUC57_mini_Ptat_AtzR) are extracted and purified together to be transformed into DH5a to obtain the target bacteria C. After screening by two antibiotics (kana&amp) medium, positive transformant - bacteria C would be further cultured and used to detect cyanuric acid.

Cell-free gene expression (CFE) has recently emerged as a powerful strategy for rapid, field-deployable diagnostics for nucleic acids and chemical contaminants.One reason for this success is that CFE reactions minimize many of the constraints of whole-cell sensors, including mass transfer barriers, cytotoxicity, genetic instability, plasmid loss, and the need for biocontainment. In addition, CFE reactions can be stabilized through freeze-drying and then are activated upon rehydration, enabling the biosensors to be used outside the laboratory at the point of sampling in the field.

Therefore, we would introduce the concept of CFE into our biosensor in order to produce a portable CFE Atrazine biosensor kit that could be easily used by normal people in daily life outside the laboratory.

III. Expected results

1. Construct the gene system that can detect cyanuric acid.

2. Design cell-free Atrazine biosensor, and test its function.

Reference

1. Zhang X, Huang Q, Zhao ZZ, Xu X, Li S, Yin H, Li L, Zhang J, Wang R. An Eco- and User-Friendly Herbicide. J Agric Food Chem. 2019 Jul 17;67(28):7783-7792. doi: 10.1021/acs.jafc.9b00764. Epub 2019 Jul 3. PMID: 31267752.

2. Zhu M, Wang L, Wang Y, Zhou J, Ding J, Li W, Xin Y, Fan S, Wang Z, Wang Y. Biointeractions of Herbicide Atrazine with Human Serum Albumin: UV-Vis, Fluorescence and Circular Dichroism Approaches. Int J Environ Res Public Health. 2018 Jan 11;15(1):116. doi: 10.3390/ijerph15010116. PMID: 29324720; PMCID: PMC5800215.

3. Silverman, Adam D., et al. "Deconstructing cell-free extract preparation for in vitro activation of transcriptional genetic circuitry." ACS synthetic biology 8.2 (2018): 403-414.

4. 1.Liu, Xiangyang, et al. "Design of a transcriptional biosensor for the portable, on-demand detection of cyanuric acid." ACS synthetic biology 9.1 (2019): 84-94.