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. 2020 Sep 21;10:15398. doi: 10.1038/s41598-020-72387-8

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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 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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Testing leakiness of ectopic expression constructs with Flp recombinase. (A) Diagram of the approach used to test leakiness in gene expression with Flp recombinase. Wildtype V. cholerae cells have intact lacZ and form blue colonies on X-gal plates (top). Cells with lacZ::FRT-Ab^R-FRT have inactive LacZ and are white on X-gal plates (middle). Flp recombination resolves the FRT-Ab^R-FRT cassette within lacZ (making lacZ::FRT), which restores LacZ activity (bottom). (B) Single colonies of cells harboring the indicated ectopic expression constructs integrated at the VCA0692 locus and lacZ::FRT-Ab^R-FRT were grown in LB medium without any inducer overnight. Then, % resolution of lacZ::FRT-Ab^R-FRT was determined by plating for quantitative culture on X-gal plates. Percent resolution is defined as the number of blue colonies/total CFUs. Data in (B) are from 6 independent biological replicates and shown as the mean ± SD. Statistical comparisons were made by one-way ANOVA with Tukey’s post-test. LOD, limit of detection, ***P < 0.001.