Laboratory Experiments Overview

Several laboratory experiments were conducted since the 4th of August 2025 in different laboratories depending on the experiment type. They are described in various wiki pages and summarized below:

A. Detection experiments

  • Detection experiments were conducted by Hugo Dupuis, Romain Héraud and Jean Schmitt in the CIRI lab between 04/08/25 and 07/10/25: Detection CIRI notebook.

    • We transformed E. coli MG1655 ΔRM with a reporter system composed of a PFOA sensitive promoter linked to an RFP reporter, enabling the detection of a PFOA-dependent fluorescent signal.

  • Another part of the detection experiments was conducted in the Lyon 1 MAP laboratory by Véronique Utzinger between the 15/09/25-26/09/25: Detection MAP notebook.

    • E. coli DH5α was transformed with a construct we designed to enhance the specificity of a biosensor system. In this design, a detectable signal is produced only if two promoters are activated simultaneously, thereby improving the specificity and robustness of a biosensor that relies on promoter activation in response to a given stimulus.

B. Degradation

  • In the Lyon 1 MAP laboratory:
    • By Matthieu Andriamisetra and Waliya Basson Hadjeris between 04/08/25 and 29/08/25 : Notebook Degradation 1.

      • This notebook compiles the experiments carried out during August 2025 as part of the cloning and fluorescent vesicle production project. It summarizes, week by week, the objectives, methods, results, and key observations. Protocol optimization was performed using a fluorescent control construct to compare two plasmid backbones, pLR81 and pIBA37. This control allowed monitoring of protein production dynamics, assessment of expression profiles, and visualization of vesicle formation, while also facilitating the tracking of downstream steps such as filtration and purification.

      Cloning and transformations were first performed in E. coli DH5α for preliminary validation. For protein overexpression, several wild-type E. coli strains (BL21, HB42, and MG55) were tested, with MG55 showing the highest protein yield and ultimately being selected as the production host. This document provides a complete record of the experimental progress, including both successes and challenges, and serves as a foundation for further optimization and continuation of the project.

    • By Baptiste Clément and Jérémi Badin between 25/08/25 and 19/09/25: Notebook Degradation 2.

      • Different dehalogenase mutants as well as a lipase SpL were cloned and introduced into several bacterial strains to evaluate their expression levels. The strain showing the highest protein production was selected for subsequent experiments.

      Purification protocols based on extracellular vesicles and bacterial pellets were then tested. A Neo Green–tagged protein was used as a tracer to monitor protein losses throughout the purification steps.

      The most efficient conditions were retained for the final purification of the enzymes, yielding purified samples suitable for further functional analyses.

    • By Maëva Chopis and Elisa Afonso between 15/09/25 and 07/10/25: Notebook Degradation 3.

      • Protein purification was carried out using cell lysis by sonication combined with lysozyme, enabling the production of a substantial amount of protein (RPA1163, Lipase SpL, and one RPA1163 mutant). For testing dehalogenase activity, various colorimetric methods were evaluated to assess proton (H⁺) release, one of the reaction products, during defluorination.

  • Analyses by NMR and HPLC-UV were performed in the INSA MAP lab by Noémie Boucetta and Sarah Gaborit between 04/08/25 and 16/08/25 : Notebook Analyses.

    • Synthesis of N-[(4-chlorophenyl)methyl]-2,2,2-trifluoroacetamide (cyclic TFA), and development of an HPLC-UV method to detect both the reagent 4-chlorobenzylamine and the product N-[(4-chlorophenyl)methyl]-2,2,2-trifluoroacetamide formed in the reaction catalyzed by Lipase SpL.

C. Perspectives

Additionally, experimental perspectives on enzyme characterization within the degradation project can be found on the Perspectives page.

Furthermore, all parts are described on the Parts page.