The 5′ ends of
the forward and reverse primers were designed to be complementary to the last 80 nt of each tagged ORF, not including the stop codon, and to the 80 nt immediately downstream of the stop codon, respectively. The resulting PCR products were transformed intoSalmonellaST14028s strain carrying plasmid pKD46. The tagged mutants were constructed using the λRed recombinase method , following the procedures as described previously . The tagged mutants were selected in the presence of kanamycin and further confirmed by PCR. Table 3 The primers used to construct www.selleckchem.com/products/DMXAA(ASA404).html the tagged strains T-prgI, T-sipA, T-sipB, T-sopE2, T-spaO, and T-sptP. ORFs Upstream primer Downstream primer prgI 5′ATAACTTGTACCGTAACGCGCAA TCGAACACGGTAAAAGTCTTTAAG GATATTGATGCTGCCATTATTCAGA ACTTCCGTGATTACAAGGATGACG ACGA 3′ 5′GACCTGATATTGACCGCCTGCCC TATAACGGCATTCTCAGGGACAAT selleck chemicals llc AGTTGCAATCGACATAATCCACCTT ATAACTGACATATGAATATCCTCCT TAGTT 3′ sipA 5′GCCCGGCTTACGAGTCATACCTG AATAAACCTGGCGTGGATCGGGTT ATTACTACCGTTGATGGCTTGCACA TGCAGCGTGATTACAAGGATGACG ACGA 3′ 5′ACATCAACGGCAATACAAGAGG TGATCACTTTTTTGACTCTTGCTTCA ATATCCATATTCATCGCATCTTTCC CGGTTAACATATGAATATCCTCCTT AGTT 3′ sipB 5′CGGAACTGCAAAAAGCCATGTCT
TCTGCGGTACAGCAAAATGCGGAT GCTTCGCGTTTTATTCTGCGCCAGA GTCGCGCAGATTACAAGGATGACG ACGA 3′ 5′GAATGATTATTTAAATAAGCGGC GGGATTTATTCCCACATTACTAATT AACATATTTTTCTCCCTTTATTTTGG CAGTTTCATATGAATATCCTCCTTA GTT 3′ sopE2 5′ATAAGGGGACGATGCCAACGCC ACAACAATTTCAGTTAACTATAGA AAATATTGCGAATAAGTATCTTCAG AATGCCTCCGATTACAAGGATGAC GACGA 3′ 5′TCGCCATAAAAACGAATATATAG TTTCAGAAAATCTGCTATTAATTCA TATGGTTAATAGCAGTATTGTATTT ACTACCACATATGAATATCCTCCTT AGTT 3′ spaO 5′ATGAATGACACCTTAGGCGTTGA GATCCATGAATGGCTGAGCGAGTC TGGTAATGGGGAAGATTACAAGGA TGACGACGA 3′ 5′CCTGACGCAATAATAAATGGCAA CAGGGTGGAAAATGCCAGTAAGGC GABA Receptor AATTAATGAGATACATATGAATAT CCTCCTTAGTT 3′ sptP 5′GCCTCACAGTTTGTACAACTAAA AGCAATGCAAGCCCAGTTGCTTAT GACGACGGCAAGCGATTACAAGGA TGACGACGA 3′ 5′CAAATAATTATACAGAAATAGCT TACTTTCAGATAGTTCTAAAAGTAAG CTATGTTTTTACATATGAATATCCTC CTTAGTT 3′ Once homologous
recombination was confirmed, the tagged mutations were transduced into fresh cultures of ST14028s by transduction using phage P22, and P22-free colonies were selected, following procedures described previously [46,47]. To generate non-polar strains, each of the tagged mutants was transformed with plasmid pCP20 by electroporation. The non-polar strains were selected for their sensitivity to kanamycin and further confirmed using PCR. The regions for the tagged ORFs in all the generated tagged strains (i.e. the homologous recombinant mutants, P22-trasduced mutants, and the non-polar mutants) were sequenced to confirm that no other mutations were found in these regions. In vitrostudies of the expression of the tagged SPI-1 proteins Colonies of tagged strains were inoculated in 1 ml of LB broth and cultured at 250 RPM and 37°C for 16 hours. The bacterial cultures were used to prepare for the pellet and supernatant samples for Western analysis.