Figure 2

Overview of steps involved in chromosomal mini-Tn 7 - pir insertion. A) Introduce the mini-Tn7-pir⁺ delivery vector by conjugation. Select ampicillin resistant (Amp^r) colonies at 30°C to establish the delivery vector. B) Grow Amp^r cells at 30°C in presence of arabinose to induce the genes encoding the Tn7 site-specific transposition pathway. C) Introduce the ori_R6Kγ reporter pR6KT2 by conjugation and grow at 37°C in the presence of gentamicin (Gm) to cure the mini-Tn7-pir⁺ delivery vector and report integrants based on pir-dependent replication of pR6KT2. Establish Gm^r and Amp susceptible (Amp^s) phenotype. D) Cure pR6KT2 reporter plasmid by plating on sucrose-containing medium. Verify Gm^s and Amp^s phenotype, and confirm pir⁺ integrants by PCR (using primer pair 2372 and 2373 for E. coli or 2374 and 2375 for S. enterica serovar Typhimurium). It must be noted that PCR-based insertion site verification strategies are limited to bacteria for which sequence information about the glmS flanking sequences is available. Methods for identifying Tn7 insertion sites in bacteria for which genome sequences are unknown have been described [12].