Team:Shanghai United/Engineering
Engineering
Introduction
Arsenic is widely distributed in the natural environment. Trace amounts of arsenic can be detected in soil, water, minerals, and plants. Among many environmental pollutants, arsenic ranks first on the blacklist of polluting toxic elements and is also a powerful carcinogen. In many countries, arsenic causes water, soil and crop pollution. We design an arsenic-induced response system in the cell or on the surface of the engineered probiotic bacteria to detect the content of arsenite in the environment, provide warnings to people, and reduce the health damage it causes to the human body.
Design
Three proteins ArsR, ArsD, and ArsA belong to the operon family. The cysteine thiolates on their amino acid sequences can form a three-coordinate complex with arsenic (iii), which is self-sensing to arsenic.
Under the induction of arsenite, ArsD gene is transcript and enxpressed in E. coli cells. At the same time, a reporter gene GFP is connected to the ArsD gene. Then, when the concentration of arsenite in the environment, the expression of ArsD-GFP can be detected by observing the intensity of the fluorescent signal.
Build
Three recombinant plasmids pUC57-ArsD-amilGFP (Figure 1), pUC57-ArsR-amilGFP, and pUC57-ArsA-amilGFP were constructed in order to select the most sensitive sensor to arsenic. All the constructs are confirmed by DNA sequencing.
Figure 1. Schematic map of plasmids.
Test
Fluorescence intensity generated by an ARSD/amilGFP transformed E. coli reacting for 1 hour in C2H6AsNaO5 solutions. As seen in the figure, fluorescence was detected for all C2H6AsNaO5 concentrations that are above 0, which confirms that the designed plasmid worked as intended (Figure 2).
Figure 2: Fluorescence Intensity of Transformed E. coli In Different Concentrations of C2H6AsNaO5 Solutions
Learn
C2H6AsNaO5 was used for this function test. The observed fluorescence intensity suggested that the engineered bacteria are functional. However, the fluorescence was rather minor, so we speculate that E. coli responds poorly to organic arsenic compounds. Further experiments would be conducted to test for fluorescence intensities of E. coli in inorganic arsenic solutions.
