PCR was performed using the forward primer, 5′-ACGACAGGAAACCCTTTAGG-3′ and the reverse primer was 5′-AGCGTAATAAACAGGCACGC-3′. see more It was also cloned into a pGEM-T easy vector (Promega). The imp/ostA and msbA genes were deleted by inverse PCR, and a chloramphenicol-resistant cassette (a gift from Dr. D. E. Taylor, University of Alberta) with its coding region (from the 1-bp start codon to the 624-bp stop codon)
was then cloned into the flanking regions to replace the full-length imp/ostA or msbA gene. This plasmid was natural transformed into the wild-type NTUH-S1 strain to generate deletion mutants. Selleckchem Tariquidar chromosomal DNA of the transformants was checked by PCR with primers external and internal to the replacement site to verify the double-crossover event. Complementation of imp/ostA and msbA An imp/ostA complementation strain of NTUH-S1 was constructed as described previously [14]. The promoter site of msbA gene was predicted by using a tool available at the following website: http://www.fruitfly.org/seq_tools/promoter.html. The msbA AZD6738 molecular weight gene containing the predicted promoter region (upstream 73 bp) was obtained by PCR using the forward primer: 5′-CCAATCGCTTTAAGCTG-3′, and the reverse primer: 5′-TTAGCATTCTGTCAAACGCC-3′. Then the DNA fragment was cloned into the pGEM-T easy vector (Promega). The msbA gene with its promoter region was cut from the constructed pGEM-T easy vector and ligated
into the NruI site of the shuttle vector pHel3 (plasmid pHel3 was a gift from Dr. R. Haas, Max-Planck-Institute für Biologie, Tübingen, Germany). The constructed shuttle vector Hydroxychloroquine solubility dmso was natural transformed into an msbA deletion mutant strain to generate the msbA complementation strain. Construction of the imp/ostA and msbA double
deletion mutant The gene encoding MsbA with its upstream 458-bp and downstream 474-bp flanking region was cloned into the pGEM-T easy vector as described above. A kanamycin-resistant gene aphA-3 from Campylobacter jejuni was then cloned between the flanking regions to replace the full length msbA gene. This plasmid was natural transformed into the wild-type NTUH-S1 strain to generate the deletion mutant. Chromosomal DNA of the transformants was checked by PCR with primers external and internal to the replacement site to verify the double-crossover event. Then, chromosomal DNA from msbA deletion mutant strain (Kmr) was natural transformed into the imp/ostA deletion mutant to obtain a double deletion mutant strain. It was also confirmed by PCR with primers external and internal to the msbA gene replacement site. Southern blotting Approximately 5 μg of genomic DNA from H. pylori NTUH-S1 and the mutants was digested by Hind III and incubated at 37°C overnight for complete digestion. The digoxigenin-labeled imp/ostA and msbA probes (primers were the same as those described for slot blot) was generated by PCR.