A Protein Antibiotic in the Phage Qb Virion: Diversity in Lysis Targets Thomas G. Bernhardt, Ing-Nang Wang, Douglas K. Struck, and Ryland Youngre

Details of PCR amplification, primer design, and cloning

Primers used in this study were:

ForA2:	TCACCCGGGATCCGGGTCACCTCACACAGCAG
RevA2:	TCACCCGGGATCCAGTTTCAACGCTTTACGCG
KpnImurAFor:	AGAACAGGTACCATGGATAAATTTCGT
XbaImurARev:	GAGTGGTCTAGAGGTAGCCCC
murA-50For:	TGGGCGATTCGCCGGTAGCG
murA231For:	TAATGTATTCTGCGCACCTT
murA511For:	TGTGCTGCAACCCTGGCGGA
murA791For:	TGCTGGCGAAACTGCGTGAC
murA250Rev:	AAGGTGCGCAGAATACATTA

The last 6 primers listed were used for automated fluorescent sequencing of murA as previously described, performed in the Gene Technologies Laboratory of the Institute of Developmental and Molecular Biology at Texas A&M University, as previously described (4).

The primers for cloning of the chromosomal murA locus were KpnImurAFor and XbaImurARev. The amplification conditions were: 1 cycle at 95°C, 20 cycles of 40 s at 95°C, 40 s at 53°C, and 4 min at 72°C. To sub-clone the A₂ gene into the tac expression vector pJFlacZK (5), the A₂ gene was amplified from the plasmid pGL101(A₂⁺) (20) using the primers ForA2 and RevA2. The cycling parameters were 94°C for 30 seconds, 53°C for 30 seconds and 72°C for 2.5 minutes for a total of 25 cycles. The PCR product was digested with BamHI and ligated into appropriately digested pJFlacZK.

Supplemental Figure 1. The rat mutant is altered in a surface residue of MurA. The murA locus in pZE12-murA, pZE12-rat1 and pZE12-rat2 was sequenced using automated fluorescence sequencing and the primers listed above. The altered residue, Leu138Gln, is shown on the structure of MurA complexed with the antibiotic fosfomycin (T. Skarzynski et al., Structure 4, 1465 (1996)). PDB structure 1UAE is visualized using Cn3D (version 3.0).

Medium version | Full size version