Rebecca Whitney Bills and Dr. Bradley Bundy, Chemical Engineering Department
Introduction:
Endocrine Disrupting Chemicals (EDCs) have led to worldwide health problems, including cancers, developmental disorders, and reproductive disorders. A recent study estimates that annual health and economic costs caused by EDCs in the European Union is $200 Billion and that the costs are even greater in the US (1). This is largely due to the extensive number of undetected Endocrine Disrupting Chemicals (EDCs) that we come into contact with on a daily basis. EDCs have been found in dietary, environmental, and household chemicals. Despite this only a handful of EDCs have been characterized. The purpose of this project is to optimize a newly developed fast, inexpensive EDC biosensor, termed Rapid Adaptable Portable In-vitro Detection (RAPID) so that it can be used in human blood and urine. The project purpose of screening for EDCs in blood and urine was accomplished much quicker in the lab than we had originally planned for. This led to us finding another way we could accomplish our overall goal of optimizing our RAPID Biosensor through increasing the biosensors sensitivity and decreasing the price of our procedure.
Methodology/Results:
The first step of completing our project goal was to improve cell-free protein synthesis in urine and blood. The lab was able to accomplish this by assessing the impact of urine on pH (can impact cell-free system (2)), protein stability (urea is a natural protein denaturant found in urine (3)), and nucleic acid stability (DNA and RNA catalyze the cell-free reaction (2)). This would allow the lab the ability to see which of these three factors, pH, protein stability, or nucleic acid stability is more inhibitory for our testing and then correct for it. This allowed for the lab to engineer a better cell-free system that could be used to create a RAPID biosensor that can test for EDC’s in urine. Many of these same testing techniques were done with blood as well. This began with testing the impact of blood on pH, (blood is rich in magnesium and the magnesium concentration must be optimized for efficient cell-free protein synthesis reactions as it impacts transcription and translation enzymes (2)) and testing the nucleic acid stability (DNA and RNA catalyze the cell-free reaction (2)). After which the lab was able to come to a conclusion on which was more inhibitory for the system allowing the lab to correct for this factor and engineer a better cell-free system for the labs RAPID biosensor.
As stated in the introduction the testing on blood and urine was accomplished much quicker than we had originally planned for. Allowing us the opportunity to take our research a step further than the original proposal suggested. With the overall goal being to optimize our biosensor our lab looked at our current protocol to find new ways to improve the system. Our current goal is to increase the sensitivity and decrease the price of the biosensor through incorporating a new reporter enzyme in the biosensor protein. This was done by selecting a new substrate which is a fraction of the cost of our current substrate. When we began trying to use the new substrate in our protocol we quickly learned that a specific DNA sequence in our E. coli cells was already leading to the production of the enzyme upon which the substrate acted. This has required us to begin making a new strain of bacteria that lacks the DNA sequence which is producing the enzyme. We have attempted to accomplish this through a sequence of steps to create a lac operon knockout strain by using polymerase chain reaction (PCR) to clone thousands of copies of replacement DNA sequence and introduce it into the bacteria through electroporation. As of now we have been unsuccessful in isolating the replacement DNA and transferring it to our E. coli cells, but are hopeful that we will soon be able to accomplish this task.
Discussion:
As we continue to work with our RAPID biosensor our focus shifted to analyzing what we could do to improve our current protocol and optimize our current capabilities. We have decided to try to simultaneously increase the sensitivity and decrease the cost considerably by selecting a new substrate. Though we have run into some issues with this change we feel confident that once it is implemented we will begin to take steps towards validating our RAPID biosensor by testing different household products first for known EDCs, before moving onto testing for unknown EDCs. As we continue to research and develop our biosensor we are confident that we will find a cost-effective mechanism for assessing EDC contamination and will eventually lead to an overall improvement of health worldwide.
Conclusion:
This year’s work in the lab has led to us finding new possibilities in further developing our system of testing for EDCs at a fraction of the current cost and has helped us progress towards creating our long-term goal of a viable, “Just add sample” EDC biosensor.
References:
1. Trasande, L. et al. Estimating Burden and Disease Costs of Exposure to Endocrine-Disrupting Chemicals in the European Union. J Clin Endocrinol Metab. 2015.100:1245–55.
2. Jewett, M.C. et al. Mimicking the Escherichia coli cytoplasmic environment activates long-lived and efficient cell-free protein synthesis. Biotechnol. Bioeng.2004. 86:19-26.
3. Ipe, D.S. et al. The basics of bacteriuria: Strategies of microbes for persistence in urine. Front Cell Infect Microbiol. 2016. 6:14.