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. 2021 Nov 22;16(11):e0260288. doi: 10.1371/journal.pone.0260288

Fig 1. Construction of B. abortus S19-GFP vaccine.

Fig 1

The integration of the gfp gene in B. abortus S19 was achieved using the mini-Tn7 system through a four-parental mating strategy. (A) Schematic representation of the integration of mini-Tn7 downstream of the glmS gene in chromosome II of B. abortus S19. (B) Verification of transposition was confirmed by PCR using primers pairs shown by convergent arrows that yield PCR fragments indicated in bp. Lane 1: DNA from B. abortus S19, lane 2, DNA from S19-GFP; lane 3, water. (C) The insertion site for the mini-Tn7 in B. abortus S19 was determined to be in an intergenic region, 25 bp downstream of the glmS gene. (D) Differentiation of S19-GFP vaccine from reference Brucella strains by PCR. Mini-Tn7-GFP tagged vaccine amplified two bands (200 and 432 bp amplicons) that corresponded to the mini-Tn7 and the gfp gene. In the untagged S19 strain, a unique 328 bp band of the intergenic region within glmS and recG genes is shown. “M”, molecular weight ladder; “C-“, negative DNA control from E. coli TOP10 strain.