Team:USAFA/Description
Project Description
Our project is comprised of two main goals: detection and degradation of per- and polyfluoroalkyl substances (PFAS). PFAS are synthetic compounds produced for a wide variety of purposes, including components of firefighting foams, non-stick cookware, stain-resistant material, and outdoor waterproof coatings. The main two PFAS compounds are perfluorooctanoic acid (PFOA) and perfluorosulfanoic acid (PFOS). These very stable compounds persist and accumulate within the environment and in living organisms. There are increasing concerns that high levels of PFAS accumulation can correlate with significant health conditions such as cancer and birth defects. High concentrations of these compounds have been shown to cause cell-membrane disruption, oxidative stress, and DNA damage, but long term effects on humans are not completely understood. Current methods to test and diagnose PFAS contamination in water are limited, costly, and time consuming. Additionally, once PFAS contamination is identified, there is limited ability to break down the compound and properly dispose of it. The USAFA iGEM team is genetically engineering bacteria to detect PFOA and PFOS, with the ultimate goal of designing a bacterial biosensor that can be utilized as a field test to monitor PFAS contamination levels. In addition, the USAFA iGEM team is hoping to provide a novel bioremediation tool for PFAS degradation in the future.
Detection
The ultimate goal of our detection project is to develop a diagnostic tool that can be deployed on site, accurately and rapidly monitoring levels of the concentration of PFOA and PFOS in environmental samples. A natural soil bacteria, Rhodococcus jostii strain RHA1, has been shown to respond to PFAS contamination by activating a stress response, specifically the propane monooxygenase hydroxylase alpha (prmA) gene. The detection project is employing synthetic biology and bioengineering in order to clone the prmA gene promoter and use it to drive the expression of a fluorescent reporter protein. mRFP (BBa_E1010) was selected as the fluorescent reporter protein because of its strong expression and easy detection. The prmA promoter appeared to activate transcription and expression of the downstream gene in a dose-dependent manner and it is anticipated that the fluorescent reporter signal will be proportional to the concentration of PFAS in samples, providing a novel and accurate mechanism to detect relative abundance of PFAS contamination.
Degradation
Biological Degradation Testing
The other aspect of our project was to develop a microbial based system for degrading these per- and polyfluorinated compounds. Current methods to remediate PFAS contamination utilize expensive Granular Activated Carbon (GAC) filters that help pull the compounds out water, but the bigger problem remains with what to do with the contaminated filters. GAC is an expensive mechanism to clean up water, but does not breakdown the toxic compounds. Degradation of these PFAS compounds is difficult due to the stable nature of fluorinated compounds. Recent studies have identified bacterial strains that can break down these PFAS compounds. Our team came across two papers describing the PFOA and PFOS degrading capabilities of two unique bacteria strains of the Pseudomonas genus by Yi L, et. al. and Kwon B, et. al., respectively. We were unable to contact the PI’s of each paper to get a sample of each species, and settled with using highly similar species determined by 16S BLAST. These bacteria were from the genus Pseudomonas, and are often known as degrading species for many varieties of compounds, including halogenated compounds. After obtaining these species through ATCC , we set out to quantify PFOA and PFOS in solution after being exposed to each species.
Overall, this method failed to work. Our group attempted to replicate these results over multiple-month testing. Unfortunately, the results of these two groups were unable to be replicated. This can either be due to the difference in bacteria, method for quantification that relied on LC/MS for our lab, or other unforeseen factors. Regardless, attempts of biological degradation were unsuccessful, and a new path was decided on.
Chemical Degradation: Proof of Concept
According to a paper published by Shannon M. Mitchell, et. al., degradation of PFOA can be chemically mediated by catalyzed Hydrogen Peroxide Propagation (CHP) reactions. This reaction uses Iron (III) as a co-substrate to generate reactive ions that were claimed to disrupt the Carbon-Carbon backbone of the PFAS. This paper determined that the generated superoxide and peroxide ions generated contributed to PFOA degradation. Additionally, they determined that generated hydroxyl ions do not contribute to degradation. Our team replicated this finding with minimal changes to the procedure. We were not able to get the same specificity of the paper due to instrument restrictions; However the overall trend and results are highly similar. See the results pagefor more information. This proof of concept confirmed that PFAS are degradable by this peroxide mechanism. This chemical step served to be crucial to the future direction of PFAS degradation, as it turns our attention from bacteria-mediated degradation to a possible plant-based pathway. See Future work below for more information.
Inspiration
Per- and polyfluoroalkyl substances (PFAS) have been used in industrial applications since the 1950’s. PFAS compounds are natural water repellents and have been used in commercial applications for non-stick coatings such as Teflon®, stain-resistant material, outdoor waterproof coatings, and in fire fighting foams. As a result of the abundance of PFAS applications, PFAS compounds can be detected in a majority of the United States human population as well as global populations.These synthetic compounds are emerging contaminants in surface water, groundwater, soil, and air. They are known to remain persistent in the natural environment and have recently been reported to be dangerous to human, animal, and environmental health. The United States Air Force has used fire retardants containing PFAS since 1970 during fire fighting training exercises to extinguish petroleum fires. As a result of the use of PFAS in military training, many military bases have high levels of PFAS contamination on and surrounding their bases. More than 125 military installations have recently detected PFOA contamination. The cadets at the United States Air Force Academy (USAFA) have a strong desire to serve their nation and communities, and as USAFA’s iGEM team, it was collectively decided that the team should try a synthetic biology approach to work on the global PFAS contamination problem. The United States Air Force Academy iGEM team is applying genetic engineering approaches to develop a bacterial system that can rapidly detect PFOA and PFOS in contaminated water supplies.
Future Work
Detection
Although preliminary results show that the Prma-mRFP part is up regulated when exposed to PFOA through qRT-PCR, our team will continue this research by confirming this result, and tuning expression to generate enough RFP to be visible under fluorescent spectroscopy. This will be achieved by attempting to use a high copy origin or adding required factors to allow use in a high copy E. coli plasmid. Additionally, further research is needed to better characterize the function of our prmA-mRFP insert. Once its function is better understood, we plan to optimize the system to heighten its sensitivity to dilute concentrations of PFAS. Our end goal is to incorporate our biosensor into a field test that could be used to quantify water contamination on-site.
Degradation
After the success of the chemical degradation of PFAS, our team plans on incorporating this into iGEM's scope by creating a modified plant capable of uptaking PFAS and degrading them through the use of the plant peroxisome. In Lisa M. Colosi, et. al it was shown that a reaction very similar to the CHP reaction that employs plant peroxidase and hydrogen peroxide along with a co-substrate can be used to break the carbon backbone. We will first confirm this reaction in vitro. After confirmation, we will focus on getting the reactants to the appropriate location in plant cells. One of our team's members has done research into PFAS uptake by duckweed from the genus Lemna; However there is no current research in where or how PFAS are uptaken in plant models. Our team plans to determine if PFAS are concentrated in a specific location, followed by genetic expression studies inA. thaliana. We will be specifically looking for up regulation of a transporter upon exposure to PFAS, especially if there is evidence that PFAS are concentrated in specific plant tissues. Should we find an up regulated transporter we plan on expressing it to send PFAS to the peroxisome where we can perform in vivo experiments.