Lastly, these PmBR zeocinR KanS SmR conjugants were screened by PCR using Platinum® Pfx DNA Polymerase (Invitrogen™) with the primers P7 (5′-TTG AGC ACG ACC AAC AGC AAC GTC-3′) and P8 (5′-CCA ATG CGG TCG AAT GAT TGC C-3′), which led to the identification of the mutant strain DD503.boaA. These primers yielded a PCR product of 1.3-kb in B. pseudomallei DD503 and a larger amplicon of 1.8-kb in the mutant. The primers P9 (5′-TAT CGC AAG GTT TGG AAC AAG GCG-3′) and P10 (5′-ACG CCG AAT ACC CTT GAT AGC TG-3′) were also used to further confirm gene replacement in the B. pseudomallei mutant strain. These primers amplified selleck inhibitor DNA fragments of 5-kb in the parent strain

DD503 and of 5.5-kb in the isogenic boaA mutant. After the conjugative transfer of plasmid pKASboaAZEO into the B. mallei strain

ATCC23344, colonies shown to be PmBR, zeocinR and KanS were screened by PCR with P7 and P8 as described above to identify the mutant strain ATCC23344.boaA. Of note, the boaA genes of both isogenic mutant strains DD503.boaA and ATCC23344.boaA were amplified and sequenced in their entirety to verify proper allelic exchange and successful disruption of boaA. Construction of a boaB B. pseudomallei isogenic mutant strain The plasmid pSLboaB was digested with NheI to remove a 162-bp fragment internal to the boaB ORF, treated with the End-It™ DNA End Repair Kit and ligated with the 0.45-kb zeocinR marker to yield the construct pSLboaBZEO. This plasmid was digested with BamHI and Nutlin-3 datasheet a 6.2-kb fragment, which corresponds to the boaB ORF disrupted with the zeocinR cassette, was purified from agarose gel slices, subcloned into the suicide plasmid pKAS46 and

introduced into B. pseudomallei DD503 by conjugation as described above. Conjugants shown to be PmBR zeocinR KanS SmR were screened by PCR using Platinum® Pfx DNA Polymerase (Invitrogen™) with primers P11 (5′-AGG TGG CGAC TCA AAT AGA ACC GT-3′) and P12 (5′-GTT CGT GTT GTT GGC TAC GGC AAT-3′) to identify the mutant strain DD503.boaB. These primers amplified a PCR product of 1.7-kb in B. pseudomallei DD503 and of 2.0-kb in the mutant. The primers P13 (5′-AGG TGG CGA CTC AAA TAG AAC CGT-3′) and P10 were also used to further confirm gene replacement in the B. pseudomallei mutant strain. Suplatast tosilate These primers generated amplicons of 5.2-kb and 5.5-kb in strains DD503 and DD503.boaB, respectively. Additionally, the boaB gene of DD503.boaB was amplified and both strands of the PCR product were sequenced to verify allelic exchange. Construction of a B. pseudomallei boaA boaB double mutant strain A 0.8-kb PCR product, which corresponds to a region located within the 5′end of the B. pseudomallei DD503 boaB ORF, was amplified with Platinum® Pfx DNA Polymerase (Invitrogen™) using primers P14 (5′-CTC GGG CTC AAT AAC ATG GC-3′) and P15 (5′-CGG AAT TCC GGT TCG TGT TGT TGG CT-3′; EcoRI site underlined).