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. 2019 Jun 20;9:8915. doi: 10.1038/s41598-019-45429-z

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© The Author(s) 2019

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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Construction and evaluation of a mini-ICEHptfs4 derivative. (a) The mini-ICEHptfs4 variant (P12 [mini-RJ-cat]) was generated by marker-free deletion of genes 438 to 469 in a strain containing the P_LJ-activatable cat cassette. (b) The correct deletion of the ICEHptfs4 mid region was confirmed by PCR from genomic (g) or plasmid (p) DNA preparations with the ICE-spanning primer pair WS801/WS802 (upper panels), and rejoining of LJ and RJ after chloramphenicol selection (P12 [mini-RJ-cat]C) was examined by PCR with primers CS26 and CS27 (lower panels). The left and right panels were cropped from single gels; the corresponding full-length gels are presented in Fig. S5. (c) Equal amounts of genomic DNA of the indicated strains were analysed by qPCR with primers WS807/WS808 and probe WS809 (amplifying LJ/RJ-rejoined sequences; Fig. S3a), and the resulting C_q values were normalized to flanking regions as described in Materials and Methods. The indicated values represent average −ΔC_q values from four independent qPCR measurements with standard deviations of the mean. (d) Genotypes of transconjugants from the P12 [mini-RJ-cat]C donor to P12 or ΔICE recipients were analysed by PCR with primers WS432/WS437 (upper panel) and WS362/WS363 (lower panel).