The plasmid and the spectinomycin cassette were lost in 3/120 (2.5%) find more of the clones tested. One clone that had a deletion of the expected size by colony
PCR was designated 35000HPΔflp1-3. Lipooligosaccharide (LOS) and outer membrane proteins (OMPs) were prepared from 35000HP and 35000HPΔflp1-3 and were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, as described . The growth of parent and mutant in broth cultures were also compared. RNA isolation and Real Time PCR Bacterial RNA was prepared from mid-log phase organisms by using TRIzol Reagent (Invitrogen) according to manufacturer’s instructions. After isolation, RNA was treated twice with DNaseI (Ambion) for 1 hour at 37°C and then purified by using the RNeasy system (Qiagen). Samples were checked by Agilent analysis. After optimizing primers so that their efficiencies were greater than 95%, we examined the level of transcript expression in RNA isolated from 35000HP and 35000HPΔflp1-3. Using each bacterial RNA, and either the tadA primers (P3 and P4) (Table 2) or the tadG (P5 and P6) primers (Table 2) and SYBR Green, reactions were performed in triplicate using an ABI PRISM 7000 Sequence Detector
(Applied Biosystems). Data were expressed as fold change of tadA and tadG in the mutant relative to the parent. ACP-196 Complementation of 35000HPΔflp1-3 5-FU molecular weight To complement 35000HPΔflp1-3 in trans, the flp1, flp2 and flp3 ORFs were amplified using the P7 primer with a BamH1 linker and the P8 primer with an XhoI linker. The resulting 1.58-kb amplicon was ligated into pCR-XL-TOPO (Invitrogen, Calsbad, Calf.). MS-275 price transformants were selected on Luria-Bertani plates supplemented with kanamycin (50 μg/ml). The 1.58 kb insert was released from the vector by digestion with BamHI and XhoI, ligated into pLSSK , and then transformed
into E. coli DH5α. The plasmid was confirmed by restriction mapping and designated pJW1. H. ducreyi 35000HPΔflp1-3 was electroporated with pJW1. As controls, 35000HP and 35000HPΔflp1-3 were electroporated with pLSSK. Transformants were selected on chocolate agar plates containing streptomycin (50 μ/ml) and transformants were saved and designated 35000HPΔflp1-3(pJW1), 35000HP(pLSSK) and 35000HPΔflp1-3(pLSSK). SDS-PAGE and Western Blot Analysis Whole cell lysates were prepared from 35000HPΔflp1-3(pJW1), 35000HPΔflp1-3(pLSSK), and 35000HP(pLSSK) and subjected to SDS-PAGE as previously described . In Western Blot analysis, whole cell lysates were probed with rabbit polyclonal sera that bind to Flp1 and Flp2 (kindly provided by Eric J. Hansen) as described elsewhere . Human inoculation protocol Stocks of 35000HP and 35000HPΔflp1-3 were prepared according to the US Food and Drug Administration guidelines (BB-IND 13046).