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

Nature Protocols volume 14, pages 3126–3143 (2019)Cite this article

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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 (µMA), which allow the occurrence of mutations created by DNA replication errors (MV) and the evolution of cell fitness during MA (µMA) to be followed directly in individual cells of Escherichia coli. MV provides a quantitative characterization of the dynamics of mutation occurrences, and µMA 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 µMA 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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Fig. 1: Principles of MV and μMA experiments in E. coli.
Fig. 2: MV and μMA experiments in E. coli.
Fig. 3: Fabrication and use of the mother machine microfluidic chip.
Fig. 4: Replication errors and mutation fixation.

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Data availability

No datasets were generated or analyzed during the current study. Datasets from a related study are archived at Dryad (https://doi.org/10.5061/dryad.75625).

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Acknowledgements

This work was funded by the Agence Nationale de Recherche (grant ANR-14-CE09-0015-01 to M.E.) and by the city of Paris (program Emergences 2018 to M.E.).

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Authors and Affiliations

  1. Laboratoire Jean Perrin, UMR 8237, CNRS, Sorbonne Universités, UPMC Université Paris 06, Paris, France

    Lydia Robert, Jean Ollion & Marina Elez

  2. Micalis Institute, Institut National de la Recherche Agronomique, AgroParisTech, Université Paris-Saclay, Jouy-en-Josas, France

    Lydia Robert

  3. Institute of Systems and Synthetic Biology, UMR 8030, CNRS, Commissariat à l’Energie Atomique et aux Energies Alternatives, Genopole, Université d’Evry Val-d’Essonne, Université Paris Saclay, Evry, France

    Marina Elez

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  1. Lydia Robert
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Contributions

L.R. and M.E. developed the protocol. J.O. developed the image analysis software. L.R., M.E. and J.O. wrote the manuscript.

Corresponding authors

Correspondence to Lydia Robert or Marina Elez.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Hanna Salman and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Key reference using this protocol

Robert, L. et al. Science 359, 1283–1286 (2018): http://science.sciencemag.org/content/359/6381/

Key data used in this protocol

Robert, L. et al. Science 359, 1283–1286 (2018): http://science.sciencemag.org/content/359/6381/1283

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Robert, L., Ollion, J. & Elez, M. Real-time visualization of mutations and their fitness effects in single bacteria. Nat Protoc 14, 3126–3143 (2019). https://doi.org/10.1038/s41596-019-0215-x

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