Team:SBS NY/Description

Metal<span>-</span>hunters— Brief description

Project Background

Soil pollution, though often being underestimated because of its invisible nature, has become one of the central environmental problems in China. The issue of soil contamination is particularly threatening to China, since agriculture takes up a significant proportion of China’s economy and is correlated with the health of 14 billion people through food safety.

However, According to the State Environmental Protection Administration (SEPA),the current condition of soil pollution is more severe than people thought and is continuously deterioating. 100,000 square kilometres of China’s cultivated land have been polluted, with an additional 1,300 square kilometres covered or destroyed by solid waste and another 21,670 square kilometres being irrigated by heavy metal polluted water. In total, up to 10% of China’s cultivatable land are affected by soil pollution, mostly in economically developed areas. Furthermore, an estimated 12 million tonnes of grain are contaminated by heavy metals every year, causing direct losses of 20 billion yuan ($2.57 billion USD).

Among the various types of soil pollution, heavy metal pollution catches our attention because of these following reasons: its emergence is highly related to recent industrialization in China; it casts serious effects on public health as well as society and economy; it has now become one of the major environmental investments, but is not as highly awared among public as air pollution or sea pollution; solutions aiming at solving this issue are not yet completely studied and are still undergoing researches and improvements.

Soil pollution, though often being underestimated because of its invisible nature, has become one of the central environmental problems in China. The issue of soil contamination is particularly crucial to China, since agriculture takes up a significant proportion of China’s economy and is correlated with the health of 14 billion people through food safety.

Specifically, heavy metal contamination in soil is a genuine threat to the environment, to sustainable agriculture and food safety in such following ways:

1. It does direct harm to agriculture. Heavy metals such as Cd, Zn and Hg could be absorbed by agricultural crops through soil or polluted irrigation, causing a reduction in crop yields;

2. It is responsible for causing food safety and public health issue. Such heavy metal elements would either directly enter human body through agricultural crops or gather in organisms through the food chain and eventually being taken in by humans. Metals such as Cd does great harm to human body by forming cadmium sulfide protein and gathering in blood, which targets at kidney and liver and eventually leading to metal poisoning with severe complications;

3. Its negative effects are widespread and disables eco-sustainability. Harmful heavy metals that gathers in soil would potentially spread to other parts of the ecosystem through irrigation, groundwater penetration and the food chain, affecting not only local soil and plants but instead the whole ecosystem, including water, air and wildlife.

4. It is long acting, multi-source, complicated and widespread. Heavy metal comes closely related with industrialization, which is in rapid progress and is producing complex, crossing-over sources of pollution everyday. Such features make it quick to worsen but slow to be removed, while lacking any permanent, thorough removal methods.

Project Overview

We began with background research on soil pollution causes, conditions and already-used solutions by learning from previous research in this area and reaching out to the public. According to our gathered information, current methods for tackling heavy metal pollution in soil are majorly chemical methods and biological methods. Specifically, chemical methods include chemical inhibitors to neutralize harmful chemicals in soil or to trigue certain reactions that turn harmful substances into harmless precipitation. Biological methods, on the other hand, include strategies such as introduction of earthworm in hope of improving the self-cleaning ability of soil, usage of microorganism bacterial agent to decompose pollutive substance in soil and the introduction of inedible plants such as asplenium to absorb harmful metal elements from soil.

We also learn that one of the many difficulties in this process is the identification of pollution. While soil pollution being a widespread issue, businesses and owners of lands might not be aware of the heavy metal pollution happening in soil soon enough, since soil contamination is a relatively long acting process and there is often no visible features at first. However, as long as metal ions gather in soil, they could potentially do harm to the whole ecosystem without even being noticed. Therefore, our project aims at developing a quick-acting, non chemical involved tool to identify and visualize the level of severeness of certain harmful heavy metals in soil -- Cu, Hg, Cd and Zn.

Our team’s experiment started with the identification of merR-like regulators that could potentially mark and display fluorescence of the heavy metals in soil, if provided a link to GFP or potentially chromogenic protein. Therefore, we isolated four merR-like receptor genes (merR, cueR, cadR, and zntR) for experiment, each responding to one specific heavy metal that are responsible for soil pollution, in hope of developing a helpful, visible detection tool for heavy metals in soil. We inserted the genes into pSB1C3-GFP plasmids and then transformed plasmids into E.coli (DH5α strain) bacteria. After amplification process, we then tested the effectiveness of our bacteria in response to different types and concentrations of mixed heavy metal solution through gene expression and fluorescent measuring.

During our research and experiment process, we found a possible way to go further in tackling heavy metal in soil in addition to detecting and visualizing.Therefore, we also incorporated capB gene (a gene in bacteria that has the potential of promoting absorption of cadmium ions) into our existing cadR-GFP backbone using Gibson assembly. Then we got a cadR-capB-GFP plasmid, with cadR detecting cadmium in the environment and activating the capB gene (if detected Cd), which will enable the E.coli bacteria to absorb cadmium ions with high efficiency once expressed.

MerR-like Heavy metal inducible repressor based Biosensors

Here we present to you the way that merR-like regulators work in detecting heavy metals.

First of all, in the absence of heavy metal ions, merR-like regulators will form homodimers that will bind to pMerT promotor region, preventing RNAP from binding to the promoter region and can thus prevent transcription of the gene from taking place.

Once heavy metal ions are present, they will bind to the pMerT promoter region and replace the homodimers. Therefore, as homodimers detach from the promoter region, the initiation of gene transcription -- in our case, the transcription of the merR-GFP genes, which will glow once expressed -- is enabled.

Based on the mechanism in which merR-like regulator works, we soon designed experiments that can visualize the expression of merR-like regulators.