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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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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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.
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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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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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DOI: https://doi.org/10.1038/s41596-019-0215-x
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