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Cloning Strategy for Host-Prophage Junctions in S. maltophilia T13 Using Smp131

This study outlines a detailed cloning strategy for the host-prophage junctions in Stenotrophomonas maltophilia T13 that has been lysogenized with the Smp131 prophage. It features sketches of the circular Smp131 genome showcasing the genes surrounding the predicted attP site, as well as the host chromosome with its attB site. Additionally, a map illustrates the positions of genes post-integration of Smp131. Key PCR primers are provided, used for amplifying regions of interest following digestion with HincII and NaeI, enabling effective sequencing for comparative analysis.

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Cloning Strategy for Host-Prophage Junctions in S. maltophilia T13 Using Smp131

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  1. (A) attP orf44 orf42 orf43 orf45 orf46 × Smp131 (B) attB S. maltophilia T13 chromosome before integration (C) R3 L3 NaeI NaeI R1 L2 L1 L4 R2 orf46 orf44 orf43 orf45 tRNA-Thr orf42 attL attR HincII HincII Figure S4 Strategy for cloning the host-prophage junctions from Smp131-lysogenized S. maltophilia T13. (A) Sketch depicting the circular Smp131 genome and genes near the predicted attP site. Arrows represent the genes and predicted attP site. (B) Sketch showing the host S. maltophilia T13 chromosome and its attB site. (C) Map showing relative positions of genes after Smp131 integration into host S. maltophilia T13. Primers used in PCR were: L1; 5’-TGAAAGGTGCCATGACCACACG-3’; L2, 5’-GCGTTGCCAAGGTCAGATCGG-3’; L3; 5’-CGCATCGCACTCTAGGAAGTGAAG-3’; L4, 5’-AACTGCCAGAACCTCTGCAGTG-3’; R1, 5’-CTCTTGTCCTCGCTGTCGGT-3’; R2, 5’-TGATAGCCCTATTTTCAAGGGC-3’; R3, 5’-AGGCCCAGCAGCGCA-3’; R4, 5’-TGCCTGCCGCCAGCT-3’. S. maltophilia T13 chromosome containing prophage Smp131 was digested with HincII and NaeI. The fragments were self-ligated and the circularized DNA was then used as the templates for inverse PCR. Amplicons obtained were sequenced for comparison.

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