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. 2021 Mar 19;6:38. doi: 10.1038/s41541-021-00296-6

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Fig. 1 — Cre-lox mediated deletion of the 32.5 kb dot/icm locus (cbu1622–cbu1652). a Schematic outlining the replacement of the dot/icm locus with kan^r and lysCA cassettes. A four-step Cre-lox strategy was used. Step 1 and 2 involved inserting loxP sites 5′ and 3′ of CBU1622 and CBU1652, respectively, using homologous recombination. Step 3 involved Cre mediated recombination between loxP sites resulting in deletion of the dot/icm locus. In step 4 Cre- mediated insertion of a lysCA cassette flanked by loxP sites into the single loxP site adjacent to kan^r was conducted to aid in cloning of the mutant. The location of loxP sites is denoted. b Agarose gels showing PCR product confirmation of deletion of the dot/icm locus and clonality of the mutant. The upper panel shows PCR products using primers flanking the dot/icm locus. NMI template DNA produced no band due to the size of the expected product (41.7 kb) while NMI Δdot/icm template DNA produced a band of the expected size (9.1 kb). The middle panel depicts lack of amplification of CBU1628 via gene specific PCR of NMI Δdot/icm DNA. The bottom panel shows amplification of a 1.6 kb region of gDNA that is conserved between NMI and NMI Δdot/icm.