The issue

PFAS: new toxic chemicals

PFAS (for per- and polyfluoroalkyl substances) are a family of synthetic chemicals that have been part of our daily lives for over 60 years. Found in waterproof clothing, food packaging, non-stick coated cookware, firefighting foams… these substances are the perfect candidates for many industrial applications, as they are extremely hydrophobic, lipophobic and heat resistant.

Their exceptional chemical stability is due to the specific structure of these molecules: a carbon chain bonded to fluorine atoms, as represented on Figure 1.

Carbon chain Fluorine atom C-F bond Functional group

In the PFAS family, the length of the carbon chain can vary, from 1 for short-chain PFAS to more than 10 carbon atoms for long-chain PFAS. The more carbon, the more fluorine atoms are bonded. Fluorine is part of the halogen family, as well as chlorine, bromine or iodine. It is the most electronegative element on the periodic table. This is the feature of PFAS that confers their extreme stability. Due to the high electronegativity of fluorine, this bond is the strongest in organic chemistry and requires a considerable amount of energy to break, with a bon dissociation energy of 439 kJ/mol (O’Hagan, 2008). The chemical function at the head of the PFAS defines its nature. The most common ones are carboxylic acid (-COOH) and sulfonic acid (-SO3H) groups.

Unfortunately, their advantage comes at a cost, and PFAS are now nicknamed "forever pollutants”. Why? Because PFAS are released in the environment during their industrial production or during their use in everyday life: they are so stable that they do not break down and persist around us.
PFAS have been detected in urban areas like Toulouse, our city, but also at a global scale in remote regions like in the Arctic (Routti et al., 2019) and the Himalayas (Miner et al., 2021), as well as in groundwater (Ackerman Grunfeld et al., 2024) and wildlife blood samples (Routti et al., 2019; Routti et al., 2015; Birgersson et al., 2021) (for more information about the contamination scale, go to HP - PFAS global contamination).
PFAS are not only persistent in the environment: they also accumulate in living organisms, including humans (Pérez et al., 2013). Chronic exposure to certain PFAS has been associated with a range of adverse health effects (EFSA CONTAM Panel et al., 2020), including endocrine disruption, immunosuppression, developmental toxicity, and an increased risk of certain cancers. Their bioaccumulative nature raises serious concerns about long-term impacts on human health (for more information about health effects, go to HP - PFAS and health impacts).

Our motivation

A rising concern in France

PFAS have raised serious concerns in France for several years now. In 2025, concrete actions were taken to limit their use and the exposure of the population to these chemicals. In addition to the European rules already monitoring the use of specific PFASs (like PFOS and PFOA), the French legislation built a National Plan of Action against PFAS, which was implemented in February 2025 [12]. With this initiative, France becomes one of the first European countries planning to ban PFAS in cosmetics and textile, by January 2026.
But contamination is already all over the country, all over Europe, and PFAS production itself is still not forbidden. To understand the scale of this contamination and what it involves, the French journal Le Monde and 29 other collaborators estimated the decontamination cost of PFAS in Europe.

It stands at 100 billion euros by year

The surveillance of these contamination also revealed several hotspots in France where the health of inhabitants is now at risk. We knew as an iGEM team that we wanted to use biotechnology to act for people and for the environment. Considering the scale of PFAS pollution, our choice was made, and PFAway was born.

Our solution

PFAway: decontaminating our environment with a fully integrated solution

PFAway is a bioremediation solution born to remediate the PFAS contamination in water in three ways :

• Degrading long-chain PFAS
• Defluorinating short-chain PFAS
• Increasing the resistance of our bacterial chassis to fluoride anions

1. Degrading long-chain PFAS

Degrading long-chain PFAS is the first step of the process, using a newly described bacterium: Labrys portucalensis strain F11. This strain has the capacity of degrading three different types of eight-carbon chain PFAS into smaller chain PFAS like trifluoroacetic acid (TFA, three fluor-carbon bonds) and perfluoropropanoic acid (PFPrA, five fluor-carbon bonds) [1].

alt="Structure d'une molécule typique de PFAS">

After degradation of PFAS into TFA and PFPrA, the remaining fluor atoms are then removed by an engineered dehalogenase.

2. Defluorinating short-chain PFAS

This is the core part of PFAway: enabling the enzymatic defluorination of polyfluorinated compounds, such as PFPrA and TFA released by L. portucalensis. As a starting point, a defluorinating enzyme is used: the Delftia acidovorans dehalogenase 2 (D90423.1). This enzyme was used in several previous iGEM projects (USFA 2020, USAFA 2021, SDU Denmark 2023, Padua 2024) and described in the literature as having some activity on monofluoroacetate (MFA), difluoroacetate (DFA), and TFA [15]

As shown on the table above, the more fluor atoms, the less enzymatic activity. However, common eight-carbon chain PFASs harbor 15 fluor atoms per molecule, which is beyond the degradation ability of the wild-type enzyme. Therefore, our goal is to improve the activity of the dehalogenase so that it can be more efficient on poly-fluorinated substrates. To do so, we used an innovative directed evolution approach based on in vivo mutagenesis: orthogonal replication (see the details of this technique in Close up on orthogonal replication).

The improved dehalogenase is then expressed in Pseudomonas putida KT2440 for defluorination of short chain PFAS released by L. portucalensis.

In our project, the dehalogenase gene will be inserted in the orthogonal DNA plasmid and mutated in vivo at high rate to generate many variants that will be screened by fluorescence using a fluoride-sensitive aptamer. This technique shows several advantages for us and future iGEM teams : High variants generation rate Fast screening with fluor aptamers No risk of deleterious chromosomic mutations The dehalogenase variants displaying the best activity on TFA and/or PFPrA will then be selected for expression in P. putida. Establishing orthogonal replication in E. coli is a challenging task. We contacted the authors of the original paper to share with us some materials and tips but did not get an answer. Mindful of the broad potential of an orthogonal replication system, we nonetheless decided to implement it fully on our own and to facilitate its wide adoption by future iGEM teams by making all materials and protocols fully available.

3. Enhancing resistance to fluoride anions

Defluorination of short-chain PFAS will generate F- anions in the cells. Fluoride ions are known to be trapped in the intracellular matrix due to the weak acid accumulation effect [5] and to inhibit essential metabolic enzymes [16]. Therefore, it is critical to enhance the stress resistance of our bacterial chassis to fluoride ions, especially when it comes to degrade poly-fluorinated compounds with the engineered dehalogenase.
To this end, our chassis will be overexpressing a fluor resistance factor: FluC (UniProt : Q88FT1), a transmembrane fluoride specific transporter. This homodimer protein is the most important fluoride resistance factor in strain KT2440 [4]. This is why we chose to overexpress this transporter to enhance the cell’s tolerance to high intracellular concentrations of F- anions.