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Figure 3 | BMC Research Notes

Figure 3

From: A single copy integration vector that integrates at an engineered site on the Staphylococcus aureus chromosome

Figure 3

Integration of pLL102 into an engineered attB 2 site. Figure 3A illustrates recombination between the attP 2 site on pLL102 and the attB 2 site in the chromosome of CYL12349. The same 5 bp mutations were introduced into the bacteriophage L54a wild type attP and attB sites to construct attP 2 and attB 2. The attB 2 site was placed in the chromosome (thick solid line) of S. aureus strain RN4220 between the orfs for SAOUHSC00009 (serS) and SAOUHSC00010. Recombination between attP 2 and attB 2 results in integration of pLL102 into the chromosome as illustrated in the lower portion of the figure. The L54a Int protein is provided in trans by pYL112Δ19. Short arrows represent the location of the primers. Figure 3B, Verification of integration via PCR. Genomic DNA was isolated from CYL12376 and used in PCR reactions. Products were analyzed on 1% agarose gels. Lane 1 shows the product from PCR using primers scv8 and OU9R7. Lane 2 shows the product from using primer pairs OU9R10 and scv4. The priming sites and predicted sizes of the PCR products are shown in the lower portion of Figure 3A. Lanes 3 and 4 contain PCR reactions using primer pairs Scv1 and Scv8 and Scv4 and Scv2.1, respectively, to verify that pLL102 did not integrate into the wild type attB site that is flanked by Scv1 and Scv2.1 primer sites (not shown [2]). Integration at the wild type attB site would generate 1.3 kb and 0.7 kb fragments in lanes 3 and 4, respectively.

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