Abstract
In order to identify host components involved in the infective process of bacteriophages, we developed a wide-range strategy to obtain cell envelope mutants, using Escherichia coli W3110 and its specific phage mEp213. The strategy consisted in four steps: (1) random mutagenesis using transposon miniTn10Kmr; (2) selection of phage-resistant mutants by replica-plating; (3) electroporation of the phage-resistant mutants with mEp213 genome, followed by selection of those allowing phage development; and (4) sequencing of the transposon-disrupted genes. This strategy allowed us to distinguish the host factors related to phage development or multiplication within the cell, from those involved in phage infection at the level of the cell envelope.
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Acknowledgements
We specially thank to Dr. F. Fernández-Ramírez (Hospital General de México, SSA) for proofreading of the manuscript and Dr. M. de la Garza (CINVESTAV-IPN) for providing access to her laboratory facilities. We acknowledge M.Sc., M.A., Guadalupe Aguilar-González (CINVESTAV-IPN) for technical assistance in DNA sequencing. This work was supported by Secretaría de Ciencia, Tecnología e Innovación de la Ciudad de México (SeCyTI), grant No. PICSA11-107.
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Reyes-Cortés, R., Arguijo-Hernández, E.S., Carballo-Ontiveros, M.A., Martínez-Peñafiel, E., Kameyama, L. (2016). Random Transposon Mutagenesis for Cell-Envelope Resistant to Phage Infection. In: Hong, HJ. (eds) Bacterial Cell Wall Homeostasis. Methods in Molecular Biology, vol 1440. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3676-2_6
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DOI: https://doi.org/10.1007/978-1-4939-3676-2_6
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