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Article dans une revue

Real-time visualization of mutations and their fitness effects in single bacteria

Abstract : Mutations are the driving force of evolution and the source of important pathologies. The characterization of the dynamics and effects of mutations on fitness is therefore central to our understanding of evolution and to human health. This protocol describes how to implement two methods that we recently developed: mutation visualization (MV) and microfluidic mutation accumulation (mu MA), which allow the occurrence of mutations created by DNA replication errors (MV) and the evolution of cell fitness during MA (mu MA) to be followed directly in individual cells of Escherichia coli. MV provides a quantitative characterization of the dynamics of mutation occurrences, and mu MA allows precise estimation of the distribution of fitness effects (DFEs) of mutations. Both methods use microfluidics and time-lapse microscopy, and a fluorescent mismatch repair (MMR) MutL protein is used as a marker for nascent mutations. Here, we present a single protocol describing how to implement the MV and mu MA methods, including detailed procedures for microfluidic setup installation, data acquisition and data analysis and interpretation. Using this procedure, the microfluidic setup installation can be completed within 1 d, and automated data acquisition takes 2-4 d.
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Article dans une revue
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https://hal.inrae.fr/hal-02868799
Déposant : Pascal Lallemant <>
Soumis le : lundi 15 juin 2020 - 16:03:26
Dernière modification le : mardi 16 juin 2020 - 03:40:27

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Lydia Robert, Jean Ollion, Marina Elez. Real-time visualization of mutations and their fitness effects in single bacteria. Nature Protocols, Nature Publishing Group, 2019, 14 (11), pp.3126-3143. ⟨10.1038/s41596-019-0215-x⟩. ⟨hal-02868799⟩

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