Lead Detection

According to the WHO, 40% of the planet does not have access to clean water. Lead is a serious contaminant around the world. It can cause neurological disorders, anemia, and death. In the United States alone, 3.5 million people have high levels of lead in their blood, so the problem is prevalent in both the developed and developing world. Children under the age of 12 are most at risk for lead poisoning. For more on lead poisoning, click [http://www.aquasanastore.com/water-you_c04.html here].

(Once you click, show this image.) Lead contamination is a serious public health concern. However, existing lead sensors are too expensive and often require extensive lab tests. Home lead testing kits suffer from lack of accuracy – lead is often confused with other heavy metals, such as cadmium or copper. There is a need for inexpensive and selective lead detectors. We have decided to build a lead sensor in E. Coli, a cheap, simple solution to our problem. In our search for genes that can detect lead, we stumbled upon the gram negative bacteria Ralstonia metallidurans, which can survive in environments with high concentrations of heavy metals. This remarkable prokaryote contains a lead binding protein, PbrR691, which activates a DNA promoter, turning on transcription of a number of lead resistance genes. This lead binding protein was shown by Chen, et al. in 2005 to be ten times more selective for lead than for several other heavy metals. Other lead binding proteins that have been identified by biologists are rarely so selective for lead. Not only is PbrR691 remarkably selective, the gene also has been successfully cloned into E Coli by this research group. We were confident that we could turn the lead binding protein and its corresponding lead promoter into new BioBricks for the registry. (Chen P, Greenberg B, Taghavi S, Romano C, van der Lelie D, He C (2005) An exceptionally selective lead(II)-regulatory protein from Ralstonia metallidurans: development of a fluorescent lead(II) probe. Angew Chem Int Ed Engl 44:2715–2719)

Design

Having found a Lead Binding Protein and its corresponding Lead Promoter in the literature, we began to generate a number of ideas for our lead detector design. We began with a simple detector: The lead promoter could be placed in front of Green Fluorescent Protein, GFP. Once lead enters the cell, it activates the Lead Binding Protein, which activates our Lead Promoter, which in turn gives us some GFP output. But we realized that we could make this simple detector much more effective with an amplification circuit.

[Image:Lead Switch.jpg]

Experimental Steps

We have three experimental steps to create our final product: 1. Develop an assay to measure AHL 2. Characterize our amplifier 3. Characterize our new parts: the lead promoter and the lead binding protein [Image:Experiment.jpg]