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. 2024 Oct 3;14:22998. doi: 10.1038/s41598-024-73879-7

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© The Author(s) 2024

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — Screening of a tisB expression library. (a) Schematic representation of generation and screening of the tisB expression library. A single-stranded DNA library, representing various tisB variants, was converted into a double-stranded DNA library via PCR and cloned into expression plasmids. Plasmids were transformed into wild type MG1655 and screened with respect to growth inhibition in the presence of L-ara (0.2%) in a 96-well format. TisB variants were categorized as non-functional (blue), intermediate (green), and functional (yellow). (b) Screening of the tisB expression library. After growth analysis, results for each TisB variant were combined to generate mean growth curves (native TisB: n = 7; K12D: n = 2; K12R: n = 5; Q19D: n = 6; Q19K: n = 7; D22K: n = 4; K12D D22K: n = 2; K12L Q19D: n = 7; K12L Q19K: n = 5; K26D: n = 4; K29D: n = 3; K26D K29D: n = 4; K26L K29L: n = 2). Three biological replicates of p0SD-tisB (black) and an empty pBAD plasmid (grey) were included as controls. The individual net charge of each TisB variant (as calculated with the Prot pi Protein Tool) is represented on the top right of each plot.