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. 2020 Aug 19;10:13981. doi: 10.1038/s41598-020-70841-1

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Genomic changes observed in the T3 bacteriophage genome over the course of 30 applications of the CAVE protocol. (a) Mutation frequencies observed in the T3 mutant gene pools after 10, 20, and 30 rounds of directed evolution. Mutation-frequency bars are color-coded corresponding to their functional genome location where they occur: blue are non-coding regions, magenta are structural genes, and purple are regulatory genes. (b) Fraction of net mutation frequencies observed within structural genes, regulatory genes, or non-coding regions, combined from final evolved generations of all T3 mutants from first and second CAVE series. (c) Comparison of mutation frequency indices at round 20 of CAVE application for two different series of T3 mutagenesis. (d) Protocol for recapitulation of mutations at specific sites using phage-rebooting. (e) Results from recapitulation of mutations at several structural genes verify that such mutations confer improved stability.