The resulting 1,068-bp product was digested with EcoRI and ligated CX-5461 in vivo with EcoRI digested pEXGm5B [20] DNA to yield pPS2882. The 1.4-kb FRT-Kmr FRT cassette of pFKm4 [20] as released by digestion with XmaI and ligated between the partially XmaI-digested chromosomal DNA fragments contained in pPS2882 to create pPS2896. The pPS2896 plasmid was used to delete the wbiE region from Bp82 by allelic exchange employing previous published procedures [20, 22]. This yielded the ΔwbiE mutant Bp82.39 and the presence of the correct mutant allele was confirmed by PCR amplification of the deletion region using primers
P2368 and P2369. Sequence-defined B. pseudomallei 1026 wbi::T24 transposon insertion buy LGX818 mutants were obtained through an ongoing project. Genomic DNA purification Bacterial genomic DNA was purified with the Qiagen Gentra Puregene Gram negative Bacteria kit according to the manufacturer’s recommendations (Qiagen, Valencia, CA). Phage particles were semi-purified by HSP inhibitor polyethylene glycol precipitation as previously described [23]. Briefly, 30 g NaCl was added to 500 mL of sterile filtered B. mallei ATCC23344 liquid lysate (108 pfu/mL) and stirred continuously on ice while 50 g of polyethylene glycol 8000 (PEG) was slowly added. The mixture was then stirred
continuously overnight at 4°C. PEG-precipitated lysates were pelleted by centrifugation at 11,000xg for 15 min at 4°C and the supernatant discarded. Pellets were suspended
in 8 mL SM buffer, combined with 8 mL chloroform, vortexed vigorously for 30 s and centrifuged at 4,000xg for 15 min at 4°C. Aqueous layers were retained and extracted two additional times with chloroform to remove any remaining PEG. This concentrated phage particles approximately 10-fold. Phage DNA was purified using a modification of the protocol described by Kaslow [24]. To 3 mL total concentrated lysate, 15 μL DNase I (1 mg/mL) and 30 μL RNase A (10 mg/mL) were added and incubated at 37°C for 30 min. Then 150 μL 10% SDS, 125 μL 0.5 M Cyclin-dependent kinase 3 EDTA (pH 8.0), and 250 μL STEP buffer [0.1% SDS, 10 mM Tris–HCl (pH 7.4), 80 mM EDTA, 1 mg/mL proteinase K] were added, and the mixture incubated for 30 min at 65°C. Genomic DNA from enzymatically treated lysates was phenol + chloroform extracted. 3.5 mL TE - saturated phenol was added to enzymatically treated lysates, mixed by inversion, and centrifuged at 800xg for 5 min at room temperature. The aqueous phase was retained and extracted twice with 3.5 mL phenol + chloroform (1:1) and once with 3.5 mL chloroform. Phage genomic DNA was ethanol precipitated by adding 1.2 mL 7.5 M NH4-acetate and 4.5 mL −20°C Ethanol (96%), followed by 15 min incubation on ice.