TABLE 2.
Oligonucleotides used in this study
| Oligonucleotide | Sequence (5′ to 3′)a | Description |
|---|---|---|
| P1 | GGTACCGAGCTCGGATCCACAAACTCGTTTGGAAGTATAAGCT | Primer pair used to amplify 1.5-kb region upstream of bb0744 with 20-bp 5′ flanking region homologous to insertion site of pCR2.1-TOPO and 3′ flanking region homologous to 5′ region of PflgB-aadA |
| bb0744US-R | TTCGCCATTCGGTACCGCGTAAAAGAAATTCTCCTATAAATTTAATTATAATCTACC | |
| pflgB-SpecF-KpnI | ACGCGGTACCGAATGGCGAATGGCGCGGCC | Primer pair used to amplify PflgB-Strr cassette from pKFSS1 |
| pflgB-SpecR-KpnI | ACGCGGTACCCCACGAAGTCCCGGGAGAACCC | |
| bb0744DS-F | GACTTCGTGGGGTACCGCGTCTTTACAGTTAGATTTAATTTGTATAAATCGT | Primer pair used to amplify 1.5-kb region upstream of bb0744 with 20-bp 5′ flanking region homologous to insertion site of PflgB-aadA and 3′ flanking region homologous to 5′ region of pCR2.1-TOPO |
| P2 | CTAGATGCATGCTCGAGCGGATTTCAAAACCAATTGAAACATTGC | |
| 744KO seq1 | TTTACTTCTTGAAGAGATCCTTAATTC | Primers used for sequencing and verification of fragments and final product of p744KO and p744Comp prior to transformation into B. burgdorferi |
| 744KO seq2 | CAAAATATCTATAAAGCTTGCCTCTTG | |
| 744KOseq3 | GTAGGTCGAAAAACGGCAAA | |
| 744KOseq4 | AAAATTAACAAACTCTTGGAGCAAA | |
| 744KOseq5 | GACAATATTCATGAAAGTGATTCCA | |
| 744KOseq6 | TTCTAAAGCTTCTAGCAAAGAAAAA | |
| 744KOseq7 | CTGCAAATCTTCCAGGCTCT | |
| aadA-F seq | CCCGTCATACTTGAAGCTAGACAG | |
| aadA-R seq | ATTTGCCGACTACCTCCTTGGTG | |
| bb0744US-744-R | CGGCTCTAGCCGAATGCGCATTACTTAACTTTCTTTAAAGTATTTACATCTAAA | Primers used in conjunction with P1 and P2, respectively, to amplify fragments containing 1.5-kb region upstream of bb0744 and bb0744 and 1.5-kb region downstream of bb0744 with 20-bp 5′ and 3′ flanking regions homologous to PflgB-aacC |
| aacC-bb0744DS-F | TTCGAGCCCATAATGGATCCCTTTACAGTTAGATTTAATAATTTGTATAAATCGT | |
| flgB-aacC-F | CCTAGGTAATACCCGAGCTTCA | Primer pair used to amplify PflgB-Gentr cassette from pBSV2G |
| flgB-aacC-R | ACGCGTAAGCCGATCTCG | |
| b-Actin-F | ACGCAGAGGGAAATCGTGCGTGAC | Primer pair used for enumeration of copies of mouse β-actin via qPCR (72) |
| b-Actin-R | ACGCGGGAGGAAGAGGATGCGGCAGTG | |
| nTM17FrecA | GTGGATCTATTGTATTAGATGAGGCT | Primer pair used for enumeration of copies of B. burgdorferi recA via qPCR (38) |
| nTM17RrecA | GCCAAAGTTCTGCAACATTAACACCT | |
| 5′ BB0744 ORF | GGATCCAGAGAAGTTGATAGGGAAAAATTAAAGGAC | Primer pair used to amplify bb0744 ORF for recombinant protein expression |
| 3′ BB0744 ORF | GCGGCCGCTATTACTTAACTTTCTTTAAAGTATTTACATC |
a
Restriction sites are underlined.