TABLE 1.
Oligonucleotides and primers used for Red/ET-recombinationa
| Oligonucleotide | Sequence (5′ → 3′) |
|---|---|
| Upper-oligoI (615-628) | ATCTGACCAAAGAAAAGAACAACGTTGAGTCGGTGTTCGCCGTTAACGGCGGCCTGGTGATGATGGCGGGATCG |
| Lower-oligoI (reverse complement orientation) | TCAACTTTGTTTTCTTCGCCCGGACGATCGGCCCAGTCCTTCAAGGAAACTCAGAAGAACTCGTCAAGAAGGCG |
| Upper-oligoII (130-149) | TGGCGATGCCGTTGCTGCCGCAAGAAGTTCAGCAGCAAGGGGTGAGCGTTGGCCTGGTGATGATGGCGGGATCG |
| Lower-oligoII (reverse complement orientation) | ATGGCATCTTTCATATTCGCCGCCACGTAGTCGGAGATATCCTCCTGCGTTCAGAAGAACTCGTCAAGAAGGCG |
| Upper-oligoIII (175-194)) | TGGCGGCGAATATGAAAGATGCCATCAGCCGTACGTCGGGCGTGGGTGATGGCCTGGTGATGATGGCGGGATCG |
| Lower-oligoIII (reverse complement orientation) | TTCTGCGCTTTGATGGCGGTAATGACATCAACCGGCGTTAGCTGGAATTTTCAGAAGAACTCGTCAAGAAGGCG |
| Repair-oligo 1: rep-acrB-Phe610Ala | ACGTTGAGTCGGTGGCAGCCGTTAACGGCTTCGGCTTTGCGGGACGTGGTCAGAATACCGGTATTGCGTTCGTTTCCTTGAAGGACTGGGCCGATCGTCC |
| Repair-oligo 2: rep-acrB-Phe615Ala | ACGTTGAGTCGGTGTTCGCCGTTAACGGCGCAGGCTTTGCGGGACGTGGTCAGAATACCGGTATTGCGTTCGTTTCCTTGAAGGACTGGGCCGATCGTCC |
| Repair-oligo 3: rep-acrB-Phe617Ala | ACGTTGAGTCGGTGTTCGCCGTTAACGGCTTCGGCGCAGCGGGACGTGGTCAGAATACCGGTATTGCGTTCGTTTCCTTGAAGGACTGGGCCGATCGTCC |
| Repair-oligo 4: rep-acrB-Phe628Ala | ACGTTGAGTCGGTGTTCGCCGTTAACGGCTTCGGCTTTGCGGGACGTGGTCAGAATACCGGTATTGCGGCAGTTTCCTTGAAGGACTGGGCCGATCGTCC |
| Repair-oligo 5: rep-acrB-Phe628Phe | ACGTTGAGTCGGTGTTCGCCGTTAACGGCTTCGGCTTTGCGGGACGTGGTCAGAATACCGGTATTGCGTTTGTTTCCTTGAAGGACTGGGCCGATCGTCC |
| Repair-oligo 6: rep-acrB-Del615-617 | GAACAACGTTGAGTCGGTGTTCGCCGTTAACGGC^^^^^^^^^GCGGGACGTGGTCAGAATACCGGTATTGCGTTCGTTTCCTTGAAGGACTGGGCCGATCGTCCGGGCG |
| Repair-oligo 7: rep-acrB-Phe136Ala | GCCGCAAGAAGTTCAGCAGCAAGGGGTGAGCGTTGAGAAATCATCCAGCAGCGCACTGATGGTTGTCGGCGTTATCAACACCGATGGCACCATGACGCAGGAGGATATCTCCGACTACGTGGCGGCGA |
| Repair-oligo 8: rep-acrB-Phe178Ala | AGATGCCATCAGCCGTACGTCGGGCGTGGGTGATGTTCAGTTGGCAGGTTCACAGTACGCGATGCGTATCTGGATGAACCCGAATGAGCTGAACAAATTCCAGCTAACGCCGGTTGATGTCATTACCG |
| Forward primer for amplification of repair oligonucleotides 1-6 | ATCTGACCAAAGAAAAGAACAACGTTGAGTCGGTG |
| Reverse primer for amplification of repair oligonucleotides 1-6 | CGCCCGGACGATCGGCCCAGTCCTT |
| Check-forward primer I | CCTTCTTGCCAGATGAGGAC |
| Check-reverse primer I | GCAGTACCCAGTTCCACGAT |
| Check-forward primer II | GTGCAGATCACCCTGACCTT |
| Check-reverse primer II | CGTTCTGCGCTTTGATGG |
| Check-forward primer III | ACCATGACGCAGGAGGATA |
| Check-reverse primer III | TAAGCTGTTGGCCTTTCACC |
The upper and lower oligonucleotides include the primer sequences for amplification of the rpsL-neo cassette (indicated by italics). The 5′ parts of the oligonucleotides are homologous to the corresponding acrB regions upstream and downstream (nucleotides 1793 to 1842 and 1885 to 1934 for exchange region I, nucleotides 338 to 387 and 448 to 497 for region II, and nucleotides 473 to 522 and 583 to 632 for region III). The exchanged nucleotide triplets in the repair oligonucleotides are indicated by bold type (e.g., GTT is changed to TTT at nucleotides 1834 to 1836 in acrB). The underlined sequences in the amplification primers are the priming parts for the repair oligonucleotides, which have to be elongated. The Check-forward and Check-reverse primers are used to confirm successful exchange of the rpsL-neo cassette and to sequence the modified region of acrB (check PCR product for acrB region I, nucleotides 1685 to 2030; check PCR product for region II, nucleotides 262 to 634; check PCR product for region III, nucleotides 442 to 691).