Project Description

Brief Intro to PFAS

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Our Team is focused on per- and polyfluoroalkyl substances (PFAS), man-made fluorinated organic compounds whose widespread use, environmental persistence, and bioaccumulative behavior have led them to be dubbed “forever chemicals.” PFAS can be found in water, soil, air, and everyday products such as food packaging and non-stick cookware. This constant exposure to PFAS has adverse health consequences, including, but not limited to, increased risk of cancer, hormone interference, and high blood pressure during pregnancy. Arizona, in particular, is severely impacted by PFAS contamination of water. The Arizona Department of Environmental Quality has determined that at least 57 public water systems in Arizona contain PFAS, and that number is only expected to increase. In fact, the Tempe area alone has six public water systems with PFAS levels that far exceed the health advisory levels established by the Environmental Protection Agency.

Fig. 1 One of the few beautiful lakes of Arizona

Our Solution in Wet Lab

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To begin, our solution comprised of a single objective: create a defluorinating enzyme library that could document and even possibly predict possible DNA sequences that have potential for defluorinating activity. However, after some discussion with the 2021 USAFA team, Dr.Rittman's lab, we gained a new perspective: It is not singularly the defluorinating enzymes that are significant; the byproducts also play a role. Therefore, we developed two new objectives: develop and test an ectopic metabolic chassis for E.coli, as well as genetically alter genomic expression levels to force dependency on our ectopic metabolic pathway.

Objective One

Our first objective proved to be relatively simple in terms of lab work. Most of our time was spent reviewing literature and understanding existing comprehensive research. From this, we generated a few likely candidates, and further assessed the possibilities of variants using NCBI's BLAST.

Objective Two

Our second objective led us down a path of further literature review, before we began the cloning steps and testing of our ectopic metabolic chassis. We decided to utilize homologous enzymes from Salmonella Enterica, as they showed greater catabolic activity compared to the endogenous enzymes of E.Coli. After a few initial trials, we were able to gain further insight on experimental design; it seemed asthough our ectopic system did not confer any extra benefit. We concluded that we needed to genomically alter E.Coli such that the only enzymes really present that could perform beta oxidation are our enzymes.

Objective Three

Our third objective was to knock out endogenous E.Coli enzymes using a KO plasmid. Due to some constraints we had with designs, we decided to take a novel approach using an origin of replication free microplasmid, which contained a recombination site and antibiotic resistance. The idea was to perform all of the cloning of the plasmid in vitro, rather than in vivo using a suicide plasmid. This effort proved successful, and we were able to create a new technique, called MiKOP. The benefit of MiKOP against

Our Solution in Human Practices

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PFAS compounds have emerged as a significant threat to human health, contributing to a host of health complications, including developmental issues in children and an elevated risk of cancer. This concern is amplified by the ubiquitous sources of PFAS exposure in our daily lives, from the water we drink to the food we eat and the products we use. Our innovative E.coli-based solution, functioning either in a bio-membrane or as an additive in the activated sludge process, has the potential to completely degrade PFAS contamination, mitigating its associated health risks and offering a global blueprint for addressing PFAS pollution.

To further combat existing PFAS contamination, the ASU iGEM team also adopted a comprehensive integrated human practices approach. This multifaceted strategy included collaborating with fellow researchers, raising PFAS awareness, and exploiting the application of our findings in the context of the human microbiome.

Research Collaboration

We engaged with experts who had previously worked on PFAS-related projects, including the USAFA iGEM team and the Rittman lab. The USAFA iGEM team's success in using E. coli and dehalogenases to metabolize PFAS informed our initial approach. Further insights from the Rittman Lab sparked the idea of combining a defluorinating construct with a metabolic construct, which could offer potential enhancements to E. coli’s PFAS degradation abilities.

PFAS Awareness Initiatives

To raise awareness of PFAS, our team created an informative heatmap illustrating PFAS contamination levels in Arizona. This visual representation simplifies the public understanding of PFAS contamination in the state and aids in the advocacy for the establishment of state regulatory limits for PFAS. We also conducted an IRB-approved survey on PFAS awareness in Arizona, which revealed knowledge gaps among respondents. In response, we hosted an advocacy and awareness event targeting college students, which generated highly positive feedback and reinforced our team’s commitment to future PFAS awareness initiatives.

Human Microbiome Application

Dr. Miyeko Mana and her PhD student, Dominic Saiz, provided valuable insights into applying our PFAS degradation research to the human microbiome. They proposed understanding the competition between PFAS and usable fatty acids, potential impacts on cellular energy production, and methods to block PFAS entry into cells as potential areas of future exploration.

Future Implementation

The versatility of a bacterial bioremediation solution is indicative to the limitless potential uses. From waste water treatment and industrial waste management ,removing pollutants from soil and treating drinking water, to even the implementation of a solution in the microbiome.
There presents clear potential for a synthetic biology solution that can tackle these looming challenges.
One such potential for how our E.coli could best impact the water systems servicing our populations is the development of a bio filtration system around its capabilities. By developing this as part of a multi-stage water treatment process we can create a solution that would be easily retrofitted into a current day water treatment plant. The bacteria would be used as part of a bio-membrane where contaminated water will filter through and sequester PFAS contaminants. Not only would this remove the PFAS, but also with the Beta-FluorinX's defluorinating enzymes, it would degrade the pollutants, creating a process for removing these 'forever chemicals'. Post filtration there would be testing stations along the water filtration line looking for bacterial and chemical contaminants. This process is already done in every water treatment plant, but would now come with the added safety measure of testing for bacteria. Once the water passes through the biofilter, it would enter a UV sterilization chamber. After the sterilization process, it would pass through membrane filtration to remove any such bacteria from the water. For waste water, the Beta-FluorinX could be used as an additive in the activated sludge process in a similar way to proposed bioremediation of microplastics from waste water. By adding this bacteria to the activated sludge before it goes through the filtration and cleaning process it will be able to prevent release of PFAS into the environment. This process could be used to manage industrial waste streams as well, where Beta-FluorinX could be used to filter waste before it leaves the manufacturing/processing facility and enters public sewage and drainage systems. The multitude of uses explained above, really demonstrates why there is such a dire need for an interdisciplinary synthetic biology solution to the PFAS contamination crisis.

Overall, the ASU iGEM team's efforts extend beyond scientific research. We prioritize collaboration with both peers and experts, active work to raise PFAS awareness, and the exploration of innovative applications to address the challenges posed by PFAS contamination, particularly in the realm of the human microbiome.

Heat Map

Figure 2: A heatmap of PFAS contamination levels in Arizona in parts per trillion (ppt).

This heatmap represents preliminary data from unfinished software. Due to time constraints, we had to cut the project short and never received LC-MS testing results.