3.5. GFP Reporter System Construction

The recombinant plasmids pACYC184-F-GFP and pACYC184-C-GFP containing the Y. pseudotuberculosis ompF and ompC regulatory regions fused to GFP were constructed as previously described [7]. To obtain the coding region of GFP, the plasmid DNA pTurboGFP (Evrogen, Moscow, Russia) was digested with BamHI and HindIII (Fermentas, Waltham, MA, USA), and the resulting gfp fragment was dephosphorylated with CAP alkaline phosphatase (Fermentas, Waltham, MA, USA). The ompF/ompC promoter, and terminator regions were amplified using specific primers, which contained BamHI and HindIII restriction sites, respectively (Table S3). The resulting PCR products were digested with the same endonucleases and ligated together (separately for ompF and ompC) with gfp in a three-way ligation. Next, ligation mixtures were used as templates for amplifying ompF-gfp and ompC-gfp reporter constructions with OmpF-Bam_for/OmpF-Hind_rev or OmpC-Bam_for/OmpC-Hind_rev primers (Table S3). The PCRs were carried out using the following reagents: 2 U of GoTaq DNA polymerase (Promega, Madison, WI, USA), 1× buffer for GoTaq DNA polymerase, 0.2 mM dNTPs, 0.5 μM primers, and 25–50 ng of the template. The following reaction conditions were used: 1 cycle of 5 min at 95 °C for denaturation, 30 cycles of 20 s at 94 °C, 30 s at 55 °C, 1 min 30 s at 72 °C, and 1 final cycle of 5 min at 72 °C. The PCR fragments of expected lengths (1300–1400 bp) were cut from the agarose gel and phosphorylated with T4 polynucleotide kinase (Fermentas, Waltham, MA, USA). The resulting reporter cassettes were inserted in the dephosphorylated vector pACYC184 via EcoRV (Figure S1). Recombinant plasmids were transformed into E. coli DH5alpha strain by electroporation. The colonies were selected on LB agar plates supplemented with chloramphenicol. All constructs were verified by PCR and DNA sequencing with pACYCSal_seq and pACYCEcV_seq primers (Table S2). The final recombinant plasmids were introduced into competent cells of the Y. pseudotuberculosis 488 using electroporation.