Table 1.
Oligonucleotide primers used in this study
| Primer pair | RSa | Sequence (5′→3′)b | Application |
|---|---|---|---|
| BB0172cDNA-F | GCTTGATTTTTTTAATTTTATCC | Amplification of bb0172 500-bp fragment from B. burgdorferi cDNA | |
| BB0172cDNA-R | CGGGATTACTCCCGCCAATCCCAA | ||
| FlaBcDNA-F | AACACACCAGCATCACTTTCAGG | Amplification of flab 234-bp fragment from B. burgdorferi cDNA | |
| FlaBcDNA-R | GAGAATTAACTCCGCCTTGAGAAGG | ||
| P66cDNA-F | CAAAAAAGAAACACCCTCAGATCC | Amplification of p66 683-bp fragment from B. burgdorferi cDNA | |
| P66cDNA-R | CCTGTTTTTAAATAAATTTTTGTAGCATC | ||
| OspCcDNA-F | TATTAATGACTTTATTTTTATTTATATCT | Amplification of ospC 593-bp fragment from B. burgdorferi cDNA | |
| OspCcDNA-R | TTGATTTTAATTAAGGTTTTTTTGG | ||
| BB0172TF | NdeI | ACGCCATATGTGGGGGCAAAGAGCTGTTGAGGA | Amplification of bb0172 for cloning into pCR2.1 and pET23a for protein expression and purification |
| BB0172TR | XhoI | ACGCCTCGAGCTAAAAAGTTTCGTCCCA | |
| BB0172TM1-F | SpeI | ACGCACTAGTGGAGGACCAGGAGTTCTAATGTCAATGTTT | Amplification of the first bb0172 hydrophobic region in frame with Lep sequence |
| BB0172TM1-R | KpnI | ACGCGGTACCCCTCCTGGTCCCCAAGAAATATCCAAAAT | |
| BB0172TM2-F | SpeI | ACGCACTAGTGGAGGACCAGGAAGATATAAAATTTTTTTG | Amplification of the second bb0172 hydrophobic region in frame with Lep sequence |
| BB0172TM2-R | KpnI | ACGCGGTACCCCTCCTGGTCCTATCATCCTGACAAACAA | |
| BB0172-T | ACAGATGGCGATGATTGGGGGGAA | Sequencing | |
| SP6 | ATTTAGGTGACACTATAG | Sequencing | |
| BB0172 SDM MIDAS-1F | GAGAATTTCTTTTATTTTTGCTATTTCTCGTAGCATG | Site-directed mutagenesis of the first amino acid of the MIDAS motif, DXSXS→AXSXS | |
| BB0172 SDM MIDAS-1R | CATGCTACGAGAAATAGCAAAAATAAAAGAAATTCTC | ||
| BB0172 SDM MIDAS-2F | GAGAATTTCTTTTATTTTTGATATTGCTCGTAGCATGTTAAG | Site-directed mutagenesis of the second amino acid of the MIDAS motif, DXSXS→DXAXS | |
| BB0172 SDM MIDAS-2R | CTTAACATGCTACGAGCAATATCAAAAATAAAAGAAATTCTC | ||
| BB0172 SDM MIDAS-3F | GAGAATTTCTTTTATTTTTGATATTTCTCGTGCTATGTTAAGTGTAGATG | Site-directed mutagenesis of the third amino acid of the MIDAS motif, DXSXS→DXSXA | |
| BB0172 SDM MIDAS-3R | CATCTACACTTAACATAGCACGAGAAATATCAAAAATAAAAGAAATTCTC | ||
| BB0172 SDM MIDAS-3X-F | GAGAATTTCTTTTATTTTTGCTATTGCTCGTGCTATGTTAAGTGTAGATG | Site-directed mutagenesis of the three amino acids of the MIDAS motif, DXSXS→AXAXA | |
| BB0172 SDM MIDAS-3X-R | CATCTACACTTAACATAGCACGAGCAATAGCAAAAATAAAAGAAATTCT |
a
Restriction sites (RS), when present, are indicated by bold text in the sequence.
b
Site-directed mutations are underlined in the sequence.