| Toulouse-INSA-UT

On this page, you can explore all the contributions our team made for future iGEMers! We aimed to contribute at every level of an iGEM project, starting with parts, HP, modeling, entrepreneurship, and even software.

We adapted FluorMango, a fluorescent RNA-based biosensor (Husser et al., 2023) designed to detect fluoride ions released during the defluorination of short-chain PFAS by the enzyme DeHa2. The sensor is based on the Mango RNA aptamer, which emits fluorescence when it binds fluoride ions, allowing us to directly measure enzyme activity through changes in fluorescence intensity. We characterized FluorMango in microplate assays and droplet-based microfluidics, demonstrating its strong response to fluoride and its compatibility with high-throughput enzyme screening. This work provides a ready-to-use platform for future teams aiming to monitor enzymatic activity or detect fluoride in diverse biological and environmental contexts.
Find out about FluorMango’s coding sequence here.

Our kinetic Model bridges experimental data with computational predictions and demonstrates the feasibility of our PFAS degradation process. Future teams working on growth kinetics of microorganisms, including in co-culture conditions, can use this model to test feasibility in silico before laboratory implementation, set quantitative targets for enzyme engineering, and optimize experimental conditions more efficiently. By providing both the model structure (ODEs and reaction network, see Model page) and calibrated parameter values where available, we enable future teams to build upon our work, adapt it to their own systems, and accelerate their design-build-test cycles.

Our project aimed at enhancing Pseudomonas putida KT2440’s resistance to the toxic fluoride ions released during the bioremediation process. We created pSEVA438-fluC, a biobrick for the constitutive overexpression of FluC, a fluoride-specific transporter. We proved that this part facilitates the cellular adaptation of P. putida to high fluoride concentrations (see Results page), and is therefore relevant for future iGEMers working on PFAS degradation with a bacterial chassis.
Learn more about pSEVA438-fluC on the Materials page.

We intended to bring the recently developed orthogonal replication mechanism in E. coli (Tian et al., 2024) to the iGEM world. We managed to construct the two essential components of this new system designed for the directed evolution of DeHa2: a template DNA containing the four genes of the orthogonal replication machinery and a cloned synthetic replicon consisting of the evolvable gene (here the gene of DeHa2) and a selection marker. This material is available upon request.

With PFAway, we designed ORep for evolving the dehalogenase DeHa2. Its gene was strategically introduced in the synthetic replicon and flanked by restriction sites. Additionally, we created a biobrick in which this target gene was removed using these two sites, allowing iGEM teams to easily insert any gene of interest and apply ORep to the directed evolution of their own protein. To maximize accessibility, we provide a kit consisting of a plasmid carrying the linear replicon devoid of the gene of interest and the orthogonal replication operon. A comprehensive User’s Guide to Orthogonal Replication is included, explaining step-by-step how to construct and extract the linear replicon for mutagenesis and how to generate the orthogonal replication chassis.

Throughout PFAway, we collected a large amount of growth-kinetics data across multiple conditions, including different media, molecules, and strains. All preprocessing and analysis were performed offline in R, where we normalized the time points to account for different acquisition intervals from two microplate readers, estimated the maximum growth rate and lag time, aggregated the replicates, and generated the files used by the R Shiny app. This app is a visualization-only tool that reads the processed XLSX tables and renders interactive plots. See the Kinetic Viewer here.
To help future iGEM teams looking for a user-friendly and effective way to analyze and visualize their data, we made the R code available on GitHub (KineticAnalysisWorkflow.R) and developed a simplified app that anyone can test (AppVisualisation). See IGEM-Toulouse github.

Our team was strongly motivated to build a project with a significant environmental impact. We rapidly focused on bioremediation, and explored multiple directions and aspects to define what could be an impactful and meaningful project. This was a complex journey, and to support future iGEMers motivated to leverage biology’s powers, we decided to share our decision-making pathway on how to optimize a bioremediation project. It is compiled in our Bioremediation Project Guide.

The path to PFAS decontamination is still long. In the meantime, it is important for people to be aware of this threat to human health and to know how to identify contaminated sources. This is why we decided to share a summary of the current contamination levels (see Human Practices page), explaining why PFAS pollution is a serious issue for both the environment and human health.
We also created a book for children, featuring our mascot PFrog, to illustrate the effects of pollution on wildlife and ecosystems. This book can help raise awareness about pollution in general, not just PFAS, as the lessons apply to all types of pollutants.
Finally, a survey conducted on more than 280 respondents allowed to raise awareness on PFAS contamination and bioremediation solutions. The broad variety of profiles (age, sex and education level) of the respondents assured that our initiative reached a broad spectrum of the population. The survey not only revealed gaps in public knowledge about PFAS and bioremediation, but also highlighted people’s concerns, hopes, and willingness to support sustainable solutions. By listening to the community, we learned how to communicate more clearly, build trust, and design a project that truly responds to societal needs, showing that science and citizens can work together.