Table 2. Oligonucleotides primers used in this study.
| Gene Targeted | Primera | Sequenceb (5′→3′) |
| Gene deletion | ||
| dam | dam::Cm (F) | TTCTCCACAGCCGGAGAAGGTGTAATTAGTTAGTCAGCATGTGTGTAGGCTGGAGCTGCTTC |
| dam::Cm (R) | GGCAATCAAATACTGTTTCATCCGCTTCTCCTTGAGAATTACATATGAATATCCTCCTTAG | |
| pmrA | pmrA: (F) | GCCGCAGATGATATTCTGCAACCGTGCAGGAGACTAAGCGAATAAGTGTAGGCTGGAGCTGCTTCG |
| pmrA:: (R) | GAAGGGTCATCGCTCTTCGCTGAAAACGCATCAGGCTCACCATATGAATATCCTCCTTAG | |
| rcsB | rcsB::Km (F) | CCTACGTCAAAAGCTTGCTGTAGCAAGGTAGCCCAATACAGTGTAGGCTGGAGCTGCTTCG |
| rcsB::Km (R) | ATAAGCGTAGCGCCATCAGGCTGGGTAACGTAAAAGTGATTTACATATGAATATCCTCCTTAG | |
| Gene epitope tagging | ||
| pmrA | pmrA-3×FLAG-5′ | TCGCGGGTTTGGCTACATGCTGGTTGCCACTGAGGAAAGCGACTACAAAGACCATGACGGT |
| pmrA-3×FLAG-3′ | GAAGGGTCATCGCTCTTCGCTGAAAACGCATCAGGCTCACCATATGAATATCCTCCTTAG | |
| rcsB | rcsB-3×FLAG-5′ | CTATCTCTCTTCCGTCACCCTGAGTCCGACAGACAAAGAAGACTACAAAGACCATGACGGT |
| rcsB-3×FLAG-3′ | ATAAGCGTAGCGCCATCAGGCTGGGTAACGTAAAAGTGATCATATGAATATCCTCCTTAG | |
| Gene cloning | ||
| pmrA | pmrA-F | GATCGAATTCATGAAGATACTGATTGTTGAAGACGAC |
| pmrA-R | GATCGAATTCTTAGCTTTCCTCAGTGGCAACC | |
| rcsB | rcsB-F | GATCGAATTCCATGAACAATATGAACGTAATTATTG |
| rcsB-R | GATCGAATTCTTATTCTTTGTCTGTCGGACTC | |
| Verification of predicted construction | ||
| dam | Rpe | TACGACAACCTGAACGGTTG |
| damX | GCAGCGTGCGGTCAACATG | |
| pmrA | pmrB | CCTGCTCGAACAATTGGATT |
| yjdB | AAAAACATGTCCCGATGCTC | |
| rcsB | yojN | AGAGGTTGTATACTGAGGCGGC |
| rcsC | CTGGCGGAAGAGAAACAACG | |
| pUC18 | downlacz18 | CGTCAGCGGGTGTTGGCGG |
| Real-time PCR | ||
| 16S rRNA gen | q-16s-F | GCCGCAAGGTTAAAACTCAA |
| q-16s-R | AAGGCACCAATCCATCTCTG | |
| rcsB | q-rcsB-F | ACCGCAGCATTAAGACCATC |
| q-rcsB-R | CTCAGGGTGACGGAAGAGAG | |
| pmrA | q-pmrA-F | AACCAGCATGTAGCCAAACC |
| q-pmrA-R | AACCCTCGACCAACACTCTG | |
| wzz | q-wzz-F | CGTCGCTTCGTTCTGTATCA |
| q-wzz-R | AGGATGTTACCCAGGACACG | |
Primers were purchased from Invitrogen Inc. and were designed according to the DNA sequence information available for the S. Enteritidis strain (Salmonella spp. comparative sequencing blast server BLAST Server Database at www.sanger.ac.uk).
a
F, forward primer; R, reverse primer.
b
Underlined nucleotides indicate the sequence homologous to pKD3, pKD4 or pSUB11. Underlined and italicized nucleotidic regions indicate the restriction endonuclease enzyme cut sites (EcoRI)) incorporated into the primer sequence.