Development and utilization of new O2-independent bioreporters

• Published in: bioRxiv
• Main Authors: Agranier, Eva, Crétin, Pauline, Joublin-Delavat, Aurélie, Veillard, Léa, Touahri, Katia, Delavat, François
• Format: Paper
• Language: English
• Published: Cold Spring Harbor Cold Spring Harbor Laboratory Press 08.11.2023; Cold Spring Harbor Laboratory
• Edition: 1.1
• Subjects: Anaerobic bacteria; Anaerobic conditions; Circuits; Flavin; Fluorescence; Gene regulation; Green fluorescent protein; Host range; Molecular Biology; Plasmids; Proteins; bioreporter; Flavin-binding Fluorescent Protein; anaerobic condition; anoxia
• ISSN: 2692-8205; 2692-8205
• DOI: 10.1101/2023.11.07.566077

Fluorescent proteins have revolutionized science since their discovery in 1962. They have enabled imaging experiments to decipher the function of proteins, cells and organisms, as well as gene regulation. GFP and all its derivatives are now standard tools in cell biology, immunology, molecular biology and microbiology laboratories around the world. A common feature of these proteins is their O2-dependent maturation allowing fluorescence, which precludes their use in anoxic contexts. In this work, we report the development and in cellulo characterization of genetic circuits encoding the O2-independent KOFP-7 protein, a flavin-binding fluorescent protein. We have optimized the genetic circuit for high bacterial fluorescence at population and single-cell level, implemented this circuit in various plasmids differing in host range, and quantified their fluorescence under both aerobic and anaerobic conditions. Finally, we showed that KOFP-7 based constructions can be used to produce fluorescing cells of V. diazotrophicus, a facultative anaerobe, demonstrating the usefulness of the genetic circuits for various anaerobic bacteria. These genetic circuits can thus be modified at will, both to solve basic and applied research questions, opening a highway to shed light on the obscure anaerobic world.