Team:USAFA/Poster

Welcome to the USAFA iGEM 2020 (virtual) Poster. If you click on the words on the picture, you will get a pop-out of that section to the right. Enjoy!

Poster: USAFA



Detection and Degradation of Perfluoroalkyl Substances through Bioengineering
Jackson D. Harris1, Annelise N. Holland1, Thuytien Pham1, Brian G. Swicegood1, Alissa M. Till1, Madeline M. Reicher1, Peter G. Lochmaier1, Meaghan T. Raab1, Christopher S. Jeon1, Megan E. Doherty1, Abigail E. Loesch1, Eamon A. McHugh1, Olivia M. Orahood1, Conley L. Walters1, Anthony R. Arment1, Vanessa Varalejay2, Chia Hung2, Nancy Kelley-Loughnane2, John C. Sitko1 , Erin A. Almand1, J. Jordan Steel1

1Air Force Academy, United States Air Force Academy, Colorado 80840; 2Soft Matter Materials Branch, Materials and Manufacturing Directorate, Air Force Research Laboratory, Wright-Patterson AFB, Ohio

Per- and polyfluoroalkyl substances (PFAS) contaminate public ground and surface waters, posing serious threats to wildlife and human health. Despite the ubiquitous nature of these compounds, there are limited technologies available to both detect and degrade these chemicals. To address this urgent need, the US Air Force Academy iGEM team engineered a novel PFAS responsive promoter to act as an efficient bioreporter for rapid detection of PFAS. Concurrently, the team screened PFAS-laden soil samples and identified several microbes that survive in high concentrations of PFAS. Delftia acidovorans, one of the microbes identified, contains the genes for several dehalogenases with potential activity to break down PFAS compounds. Alternate vectors and organisms for dehalogenase expression are being explored to determine maximum efficiency at removing fluorine ions from the PFAS carbon-fluorine backbone. Collaboration with water treatment experts and military research labs provides a multi-faceted attack on the PFAS issue.
Introduction
PFAS stands for Per and Polyflourinated Alky Substances. Composed of a carbon chain surrounded by fluorines, PFAS is incredibly hard to breakdown as the tight carbon fluorine bonds are incredibly heat resistant, and the steric interactions caused by the poly fluorination makes PFAS resistant to natural degradation. In addition to their hardy nature, PFAS have been found to be very toxic to both humans and the environment. Popularized in the 1960’s, PFAS has been incorporated into many products and plastics due to its utility as an industrial surfactant. In addition, it works well in firefighting foams and has been commonly used to fight fires. The waste from these firefighting foams is often left unchecked to be absorbed into the environment and groundwater. PFAS pose a threat to both our environment and our communities.
Inspiration
What inspired your team? What motivated you to work on this particular project?
Idea
How are you going to solve the problem? Where did the idea come from?
Engineering
A library of various soil microbes were isolated from PFAS contaminated sites, which revealed Delftia acidovorans as a potential PFAS degrading strain. Dehalogenases were identified in the genome, and biobricks were generated to express these genes in E. coli for future in vitro studies. These dehalogenases are currently being investigated for defluorinating potential, and initial modeling and experimental data suggests partial defluorination. Additionally, initial observations of fungi isolated from PFAS spiked winogradsky columns suggest that fungal exoenzymes may be an additional line of research for the team to focus on.
Results
Results
Two D. acidovorans dehalogenases were cloned into and expressed by E. coli. A soluble protein lysate was extracted and then treated with fluorinated compounds PFOA or ethylfluoroacetate (EtFA; positive control). Fluoride was measured by selective ion probe (SIP) to determine if the dehalogenases catalyze the defluorination of the substrates. (Figure) From this it is seen that partial defluorination is occuring, although the equilibration point is very low. We are currently working to provide a fluoride sink in the reaction system, so that the forward (defluorination) reaction is much more favored and maximal defluorination can be determined. Figure: Dehalogenase 1, variant 2 (BBa_K3347005) soluble protein extract treated with 500 PPM PFOA or EtFA. Fluoride treated by SIP.
Human Practices
The USAFA iGEM team worked diligently to continue Human Practices efforts despite the COVID-19 pandemic. Through virtual communications, the team was able to connect with experts as well as our community to receive guidance on our project as well as educate those around us. Our team spoke with civil engineers and an Air Force firefighter to learn more about the impacts of PFAS within our communities, and more specifically, in water treatment plants and military operations. In order to educate our community while mitigating the risks of COVID-19, the USAFA iGEM team focused on telecommunications via science experiment videos and informational coloring pages. Our COOL Science Festival video and coloring pages allowed us to inform our local community as well anyone else who has access to our website, thus allowing a larger-scale impact of our research. Before the COVID-19 pandemic, the team was also able to work as judges for a local protein modeling competition. This opportunity provided the team with a platform to interact and teach young students within our community. Through our Human Practices efforts, the USAFA iGEM team has had a widespread effect on exposing the dangers of PFAS.
Results
Two D. acidovorans dehalogenases were cloned into and expressed by E. coli. A soluble protein lysate was extracted and then treated with fluorinated compounds PFOA or ethylfluoroacetate (EtFA; positive control). Fluoride was measured by selective ion probe (SIP) to determine if the dehalogenases catalyze the defluorination of the substrates. (Figure) From this it is seen that partial defluorination is occuring, although the equilibration point is very low. We are currently working to provide a fluoride sink in the reaction system, so that the forward (defluorination) reaction is much more favored and maximal defluorination can be determined. Figure: Dehalogenase 1, variant 2 (BBa_K3347005) soluble protein extract treated with 500 PPM PFOA or EtFA. Fluoride treated by SIP.
References and Acknowledgements
If not already cited in other sections of your poster, what literature sources did you reference on this poster? Who helped or advised you?