Conner Earl and Bradley Bundy, Department of Chemical Engineering Department
*Note: this report contains proprietary information and should not be circulated publicly until the final research report is accepted and published in a scientific journal. (one year)
Endocrine Disrupting Chemicals (EDCs) present problems worldwide and have been linked to cancer, developmental disorders, and other epigenetic complications. Many of these complications could be mitigated if not eradicated if these chemicals were better characterized and detectable, however an inexpensive, reliable and field deployable sensor for EDCs has yet to be developed. The purpose of this project was to explore the idea of an engineered Cell Free biosensor system that is an economic, viable method for the rapid detection of EDCs.
In collaboration with the lab of Dr. David Wood (Ohio State University), Dr. Bundy’s Biotechnology lab has already shown that fusion proteins can be used in Cell Free Protein Synthesis (CFPS) to assay EDC activity. One issue that limits the growth of this technology is the cost associated with expensive reagents used in testing. The original goal of my ORCA project was to use a new type of fusion protein that relied on the color change activity of the well characterized Green Fluorescent Protein (GFP). GFP has been utilized by scientists for years and is characterized well by its ability to glow green at certain wavelengths of light. By using this well understood, well characterized protein to detect EDC activity, the biosensor technology that currently exists could be performed at a fraction of the cost.
The first step of the project was to express the biosensor protein in a cell free system. Using a plasmid DNA template provided by the Wood Lab, the DNA was first amplified and then introduced to the cell free system in order to produce our protein. With my mentor’s help, and after many trial and error experiments, I was able to successfully produce the constructed GFP fusion protein in our system. In order to effectively detect our target EDC, however, we needed a measurable color change to occur in the presence of the EDC. We realized that we were unable to measure this color shift above the background noise of our detection system. After months of adjusting the parameters of our experiment we decided to essentially “go back to the drawing board.” We realized, upon sequencing the DNA we were using to create our protein, that the protein we were actually making had one amino acid different then what we intended to make and that this slight change was likely the cause of our problems.
As we continue to work out this issue with our collaboration with Dr. Wood’s lab, our focus was shifted to further developing and expanding the capabilities of the existing fusion biosensor protein. The capabilities we are currently working to expand include the detection of EDCs in complex mixtures such as blood, urine, pond water, and even sewage. We are also spending time testing the linker proteins within the multi-domain biosensor protein itself as well as expanding capabilities to different endocrine receptors important to physiology in the human body. Further research and development may lead to a cost-effective solution for assessing EDC contamination in a variety of circumstances and lead to an overall improvement of health and preventative measures.
The functionality of this system has allowed us to develop further analysis of EDCs in complex mixtures for a fraction of previous cost (~$0.10/sample test), allowing for further characterization of our system and progress towards our long-term motivation of a viable “Just add sample” EDC sensor.
1-Schapaugh, A. W. et al. Analysis of EPA’s endocrine screening battery and recommendations for further review. Regulatory Toxicology and Pharmacology 72, 552-561, doi: http://dx.doi.org/10.1016/j.yrtph.2015.05.028 (2015).
2- Izabela Gierach, Jingjing Li, Wan-Yi Wu, Gary J. Grover, David W. Wood, Bacterial biosensors for screening isoform-selective ligands for human thyroid receptors α-1 and β-1, FEBS Open Bio, Volume 2, 2012, Pages 247-253, ISSN 2211-5463, http://dx.doi.org/10.1016/j.fob.2012.08.002.