Team:USAFA/Design

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Project Design

Currently, there are limited efficient and low-cost methods to detect and degrade PFAS. Despite the existing shortfalls, the ubiquitous nature of microbes in these harsh environments provide multiple opportunities to take capabilities existing in nature, and use synthetic biology to adapt them to tackle this issue. We hoped to find ways to more efficiently detect PFAS and ultimately to degrade PFAS. We named our project D.R.O.P. "Detect, Remove, and Obliterate PFAS."

For detection, we tried multiple approaches. First, we improved our part from last year, further optimizing the prmA promoter to express a fluorescent protein reporter when exposed to PFAS. Second, we looked for genes and promoters that may also be increased in the presence of PFAS, improving and expanding detection capabilities.

For degradation, we screened soil samples from contaminated areas to see what natural microbes could survive in a contaminated PFAS environment. We identified microbes that can live in high concentrations of PFAS and may be able to break down the compound. This approach gave us insight into natural genes and proteins that might allow for increased efficiency in detection or better degradation. Furthermore, it provided additional protein targets for exploitation, leading to the identification of a dehalogenase gene and promoter, currently undergoing further investigation.

To characterize the microbial community from PFAS contaminated soil, we collaborated with the Air Force Research Lab and tied together metagenomics from 20+ contaminated Winogradsky columns, looking for microbes containing unique or common attributes that may contribute to a PFAS solution. With this information, models could be made for degradation or possible optimized proteins and protein expression.

Our team used an iterative approach to identify potential PFAS solutions. Microbial isolates with the potential to degrade or thrive in the presence of PFAS were identified. The genomes from potential degrading strains were studied to identify potential dehalogenase genes. Once identified, these proteins of interest were cloned and characterized to determine gene expression, in minimal media with 1000ppm glucose, 1 ppm PFOA and 1000ppm glucose, and minimal media for the positive growth control, toxicity control, and negative growth controls respectively along with minimal media and 1 ppm PFOA.

Research Approach

Create a microbiological library
Identify PFAS degrading strain(s)
Protemic Characterization
Creation of Biobricks and expression in E.coli
In vitro studies of Dehalogenase