Project Description

Without being aware of it, most of the population around the world are living under the danger of exposure to heavy metal ions.

figure 1. a model of pollution

As more and more human activities create accumulating impact, and as industries develop at an unprecedentedly rapid speed, huge amounts of heavy metal pollutants are released into the environment, contaminating the earth. Heavy metal ions such as Cadmium (Cd), Chromium (Cr), Lead (Pb), Mercury (Hg), and metalloids like Arsenic (As) cannot be degraded by organisms. As a result, these ions can accumulate, create chronic toxicity, and manifest bioaccumulation up the trophic level.

Studies show that not only is heavy metal pollution a severe issue, it is also prevalent around the world, especially in water sources. Furthermore, water contamination may accumulate in soils and sediments. In China, around 16.1% of arable land is potentially contaminated by heavy metals.

Humans then take them in without notice, either by directly drinking contaminated water or through eating other plants and animals that are previously exposed to heavy metals. If the heavy metals enriched in human bodies, they can cause serious symptoms such as diseases in the kidney, the lung, the liver, the bone, different types of cancers, and deficiency in embryonic neurodevelopment.

Current methods of monitoring heavy metals are accurate and sensitive. However, the instruments and equipment are usually expensive and difficult to operate. Therefore, most of the population is still difficult to detect the heavy metals by themselves.

Our Project

Goals:

To provide a more convenient and cheaper approach to address this problem, our team designs a whole-cell biosensor kit that can test heavy metal content conveniently and readily.

Method:

Our project's aim is to create a biosensor in order to detects the heavy metal cadmium, zinc and arsenic. For cadmium and zinc, we utilize the operon CadA to expresses reporter feruloyl esterase in Bacillus subtilis. Specifically, the CadA functions as an energy-dependent cadmium efflux ATPase and a metal-induced protein. The repressor CzrA inhibited CadA. However, Cd²⁺ competitively bind to CzrA, thus enabling the reporter feruloyl esterase to be expressed.

figure 2. The biosensor to monitor cadmium and zinc concentration

This reporter feruloyl esterase catalyzes its substrate 4-Nitrophenyl Butyrate into 4-Nitrophenol. The 4-Nitrophenol can be observed directly by eyes or at 410nm wavelength. Therefore, enzymic activity was determined at the 410nm wavelength using spectrophotometric assay.

figure 3. The enzymatic reaction of feruloyl esterase

In addition, for arsenic, our project used the ArsD promotor fused amilGFP being its reporter protein. The promotor strategy is designed based on the Shanghai_United team in 2021.

Finally, our product includes the freeze-dried biosensor in engineered E.coli or B. subtilis, as well as the substrate 4-Nitrophenyl Butyrate in PBS (phosphate-buffered-saline). The results may be observed by eye, with yellow representing cadmium and green representing arsenic in the sample. GFP representing Arsenic could also be observed in the dark due to its fluorescence.

Outlook:

This technology can be applied to populations who rely on natural water sources for living and factories which produce wastes that contain heavy metals, allowing them to better monitor heavy metal content on their own.

Reference

[1] Akcay H, Oguz A, Karapire C (2003) Study of heavy metal pollution and speciation in Buyak Menderes and Gediz river sediments. Water Res.37(4):813–822

[2] Olivares-Rieumont S, de la Rosa D, Lima L, Graham DW, D’Alessandro K, Borroto J, Martinez F, Sanchez J (2005) Assessment of heavy metal levels in Almendares River sediments—Havana City, Cuba. Water Res. 39:3945–3953

[3] Huang, Z., Liu, C., Zhao, X. et al. Risk assessment of heavy metals in the surface sediment at the drinking water source of the Xiangjiang River in South China. Environ Sci Eur 32, 23 (2020).

[4] Sodango, T. H., Li, X., Sha, J., & Bao, Z. (2018). Review of the Spatial Distribution, Source and Extent of Heavy Metal Pollution of Soil in China: Impacts and Mitigation Approaches. Journal of health & pollution, 8(17), 53–70.

[5] Moore, C. M., Gaballa, A., Hui, M., Ye, R. W., & Helmann, J. D. (2005). Genetic and physiological responses of Bacillus subtilis to metal ion stress. Molecular microbiology, 57(1), 27–40.

[6]Mastihuba, V.r., L.r. Kremnický, M. Mastihubová, et al., A spectrophotometric assay for feruloyl esterases.[J] Analytical Biochemistry, 2002. 309(1):96-101.