Upon the first stages in professional establishment, we hoped to give our biosensor to those who are familiar with environmental technology. Examples of this audience would be the Environmental Protection Agency and other environmental companies that test for DDX. After further discussion and research, we see a large need for residential testing. DDX pollution is prevalent not only in areas controlled by environmental scientists, but also in residential areas. Our implementation into this community would include testing kits with simple instructions and education about DDX in the Pine River.
Technical Challenges
Working with DDX is challenging - it is very hydrophobic. One measure of it's hydrophobic nature is the oil/water partioning coefficeint, or the K-o/w. This is a log scale measurement of how much more a molecule perfers the nonpolar oil solvent over water. For DDT, the coefficient is near 6, or about a factor of a million times more will be found in oil. This means that extracting this from the soil and mixing it with our biosensor can be a challenge. We wanted to determine the proper way to go about this - can we create a protocol, and an easy to use assay, that would allow people in the field to mix our Biosensor with DDX?
Sudan Black

Sudan Black has a predicted K-o/w over 6
In order to develop such a protocol, we first needed a molecule that can be easily tracked, yet mimics the properties of DDT. Luckily, we had the nonpolar dye Sudan Black on hand in the lab. This dye is predicted to have an even greater K-o/w than DDX, although very similar. We can monitor the presence of this dye and its solubility / dissolution by measuring the absorbance at 600nm. First, we established a standard curve in different solvent - water, Ethanol, and DMSO (as well as dilute versions, such as 7.5% DMSO). We mixed a calculated amount of soil and Sudan black dye in a serial dilution which gave different concentrations of the dye in the same amount of soil. The goal was to find out with different reagents such as ethanol and DMSO how much of the dye will be removed. After adding the reagents(solvents) to the various mixtures of soil and dye, we obtained the solutions and measured the absorbance for each one of them. This represents the concentration of the dye removed and the amount of dye retained in the soil calculated.

From these experiments, we established that 7.5% DMSO was sufficient to dissolve a great deal of the Sudan Black, approximately 10-fold greater than what would be possible with water alone. Even better, E. coli can tolerate DMSO concentrations this high. We can use this solvent, together with our low-cost filter disc assay for induction, to potentially make a cheap and rapid biosensor. It might be as easy as measuring the diameter of a circle!
To utilize our kits, we would give our users an indication of how much DDX (ppm) are present on their property. This indication will allow citizens to be more comfortable in their own homes and for businesses or residential companies and increase overall property value in Gratiot County after pollution treatment (if necessary) is completed. In order to achieve this, we would approach businesses and utilize environmental consultants as a resource to advertise our biosensor to our broad audience.
Accessibility to our community is very important to us, as Gratiot County has one of the highest rates of poverty in all of Michigan. Our biosensor is the first readily available screening tool that our area has produced at a low cost. Our test is estimated for any consumer (professional and residential) to be $20 and take six hours to be completed. Current tests are anywhere from $200-900 per sample and take up to a month to give results. Our screening protocol would give a much shorter time frame and a drastic price decrease which will then allow more funding to specific areas with high rates of DDX pollution.
Challenges that may be faced and one that we intend on addressing in the delivery of our biosensor to consumers is education. We have written a children’s book that explains DDX pollution, but contamination beyond DDX. Although written for an audience of children, this could be a useful tool in educating those who are not aware of the severity of the DDX. We also plan on hosting open panels at our college that have experts and our own student researchers. This will open a dialogue for people within the community to then provide resources for potential contamination. Safety concerns related to handling the tests properly would be addressed in both in educational sessions held by Alma iGEM and in the testing kits we plan to provide consumers.
Going forward, we plan on talking to more groups that are in Gratiot County that have a part in cleaning up the superfund site. With this, we hope to distribute our science to both professionals and residents. As of now, the superfund site is ready for residential zoning in the next two to three years. We hope to be involved in the process that includes keeping new residents safe and ensuring our synthetic biology can save our community from an ongoing tragedy.