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. 2017 Sep 26;7:12327. doi: 10.1038/s41598-017-12395-3

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© The Author(s) 2017

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 integration of a chromosomal 18-members GLOS-RedLibs ribB RBS library and FNM riboswitch removal in an MMR⁺ strain. (a) Schema of the GLOS-RedLibs ribB RBS library construction. Left: FMN riboswitch and wild type ribB RBS sequence; right: degenerate ribB RBS sequence after GLOS treatment. Note that the riboswitch is supposed to be eliminated concomitantly with the RBS modification step. (b) Library clones were analyzed by Sanger sequencing (n = 96). The 18 possible RBS sequences after genome editing (grey) as well as the wild type context (red) are listed in ascending order of their predicted TIR, except wild type since the TIR including the riboswitch cannot easily be estimated. RBS sequences showing mutations were excluded. Therefore, only 94 of 96 sequenced clones are displayed. (c) Riboflavin production of all isolated RBS library member strains (n = 96, grey, single measurements) compared to wild type (red, in triplicate, mean ± standard deviation). (d) Distribution of sequences in the oligonucleotide pool after chemical synthesis. Illumina-based oligonucleotide pool sequencing for the oligonucleotide used to construct this library. Bars in black indicate oligonucleotides for which no corresponding strains could be recovered during library-based genome editing.