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
Per- and Polyfluoroalkyl Substances (PFAS) are composed of a carbon chain saturated by fluorine atoms. PFAS is incredibly hard to breakdown. The tight carbon-fluorine bonds are incredibly heat resistant, and the steric interactions caused by the poly-fluorination makes PFAS robust against natural degradation. PFAS is highly 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. It works well in firefighting foams to suppress flames. The residual foam is often left to be absorbed into the environment and groundwater. PFAS pose a threat to both our environment and our communities. https://www.michigan.gov/documents/pfasresponse/PFAS_Cycle_Diagram_670769_7.pdf
Inspiration
The USAFA iGEM 2020 team took on inspiration from the previous USAFA 2019 iGEM project and the PFAS contamination found within the local Colorado water. Last year, the 2019 team used Rhodococcus jostii and prmA promoters to detect PFAS. This year, our iGEM team took steps to modify the promoter from last year, while searching for natural degradation methods. The USAFA 2020 iGEM team hopes to bioengineer bacteria in an effort to save our local environment and the numerous Air Force bases with PFAS contamination. A big inspiration for our project came from our cooperation with Schriever Air Force Base and the soils they contributed to our project. With over 200 unique bacterial species isolated from the Air Force Base and other contaminated regions, we created a microbiological library of potential PFAS degrading bacterium. https://www.internationalairportreview.com/article/104858/pfas-firefighting-foams-health-risk-airports/ -- https://news4sanantonio.com/news/trouble-shooters/dod-testing-for-pfas-in-water-but-not-screening-firefighters
Problem
Team USAFA is working to solve the worldwide problem of PFAS contamination. PFAS affects the world by causing metabolic disturbances in animals (Lai, K. P. et al (2018)), damaging ecosystems and contaminating our drinking water. PFAS is not naturally degradable or degradable in common water treatment systems and facilities or waste disposal sites. https://www.eea.europa.eu/themes/human/chemicals/emerging-chemical-risks-in-europe
Problem Design
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.
Parts
A total of 8 variants of dehalogenases from D. acidovorans were created and expressed in E. coli. Both unmodified and modified variants of dehalogenase type I and type II were created, with the modified variants also containing sub-variants with 6xHis tags on either N or C terminal. Modified variants (6 total) were under control of the T7 promoter for maximal protein production while the unmodified variants were expressed under control of the Lac promoter. Figure: Modeled Dehalogenase 1 from Delftia acidovorans.
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.
Future Direction
The USAFA iGEM team is currently testing purified dehalogenases against various perfluorinated compounds, including EtFA, PFOA, PFOS, and a PFAS slurry, to determine defluorination capability. Characterization of the mechanism, degradation products, speed, and efficiency will follow successful defluorination. In collaboration with the Air Force Research Lab (AFRL), genome and transcriptome sequencing will be performed to identify their changes in response to PFAS. This may elucidate a new gene of interest, warranting more focused characterization. ​ Finally, the team has recently identified a fungal species showing strong growth on solid minimal media with PFAS. A corresponding fungal exoenzyme may also be of research interest. ​

Figures: Fusarium type fungus, Penicillium sp.



Figures: Dehalogenase protein models
Acknowledgements and Literature
We thank Dr. Jordan Steel, Major Erin Almand, Captain John Sitko and Dr. Anthony R. Arment for their assistance and guidance. Additionally, Dr. David Hale and Colonel Steve Hasstedt have been constant supports for the iGEM program and Cadet Research. We would like to thank Dr. Chia Hung for guiding the molecular modeling and testing aspects of this project and AFRL.

​ Funding was provided by the US Office of the Surgeon General, the Life Science Research Center at USAFA, and the Biology Department at USAFA.​

General Support : Patti Kryzanowski, USAFA Biology Lab Manager. Morgan Vance USAFA Biology Equipment Specialist.

Project Support and Advice: USAFA Faculty: Dr. Jordan Steel, Major Erin Almand, Captain John Sitko, Dr. Anthony Arment and AFRL researchers: Dr. Vanessa Varaljay, Dr. Chia Hung, Dr. Rajiv Berry, and Dr. Nancy Kelley-Loughnane.

Fundraising/Funding Help: Dr. Don Veverka, Life Science Research Center USAFA and Dr. Nereyda Sevilla, Defense Health Agency Clinical Investigations Program.

Lab Support: Patti Kryzanowski, USAFA Biology Lab Manager.

Difficult Technique Support: Dr. Rajiv Chia Hung, Modeling Techniques.

Wiki Support: C2C Eamon McHugh.

Presentation Coaching: USAFA Biology Department Faculty.

Human Practices Support: Major Erin Almand.

Literature:
  1. Li, Y., Yue, Y., Zhang, H., Yang, Z., Wang, H., Tian, S., Wang, J., Zhang, Q., & Wang, W. (2019). Harnessing fluoroacetate dehalogenase for defluorination of fluorocarboxylic acids: in silico and in vitro approach. Environment international, 131: 104999.
  2. Sharp, J. O., Sales, C. M., LeBlanc, J. C., Liu, J., Wood, T. K., Eltis, L. D., Mohn, W. W., Alvarez-Cohen, L. (2007). An Inducible Propane Monooxygenase Is Responsible for N-Nitrosodimethylamine Degradation by Rhodococcus sp. Strain RHA1. Applied and Environmental Microbiology, 73(21), 6930–6938.
  3. Sullivan, M. (2018). Addressing Perfluorooctane sulfonate (PFOS) and perfluorooctanoic Acid (PFOA). Department of Defense, RefID: 4-0A57E2B​.
  4. Weathers,T. S., Higgins, C. P., Sharp, J. O. (2015). Enhanced Biofilm Production by a Toluene-Degrading Rhodococcus Observed after Exposure to Perfluoroalkyl Acids. Environmental Science & Technology, 49(9), 5458-5466.
  5. Yi, L. B., Chai, L. Y., Xie, Y., Peng, Q. J., Peng, Q. Z. (2016). Isolation, identification, and degradation performance of a PFOA-degrading strain. Genetics and Molecular Research : GMR, 15(2), 15028043.
  6. Zhang, X., Chen, L., Fei, X., Ma, Y., & Gao, H. (2009). Binding of PFOS to serum albumin and DNA: insight into the molecular toxicity of perfluorochemicals. BMC Molecular Biology, 10(16), 10.1186/1471-2199-10-16.