TABLE 2.
Oligonucleotide primers used to construct CTD mutation(s) in rgpBa
| Primer name | Sequence (5′→3′) | Primer location (nt)b | Description |
|---|---|---|---|
| M13 universal forward | TGTAAAACGACGGCCAGT | ||
| M13 universal reverse | CAGGAAACAGCTATGACC | ||
| dmngrForSfoI | GTCCTGCTGCCGGCGCCGAGAGCTCCCAACGCG | 2968-2978 | rgpB(nt 2986-2978)-SfoI-pGemTEasy (nt 103-118) |
| dmngrRevSfoI | CGCGTTGGGAGCTCTCGGCGCCGGCAGCAGGAC | 2978-2968 | Reverse complement of dmngrForSfoI |
| dmngrRevCM | CCCGGCAGCAGGACTTTCTAC | 2981-2961 | rgpB(nt 2981-2961) |
| dmngrForCM | GCTACTGCTAAAAACCGCATGGTATTC | 3015-3041 | rgpB(nt 3015-3041) |
| Gly699Profor | CTGACGATCTTCGATATGAACCCCCGTCGTGTAGCTAC | 2982-3019 | RgpB with Gly699 → Pro |
| Gly699Prorev | GTAGCTACACGACGGGGGTTCATATCGAAGATCGTCAG | 3019-2982 | Reverse complement of Gly699Profor |
| Gly716Profor | GAAGCACAAAACCCTGTGTATGCCGTTCG | 3042-3070 | RgpB with Gly716 → Pro |
| Gly716Prorev | CGAACGGCATACACAGGGTTTTGTGCTTC | 3070-3042 | Reverse complement of Gly716Profor |
| HSMUT2for | ATCGCTCAAGAAGGCAAGCAGTATCAAGAAGTTATAGTG | 3072-3113 | RgpB with Thr728 → Gln and Thr730 → Gln |
| HSMUT2rev | CACTATAACCTTTTCTTGATACTGCTTGCCTTCTTGAGCGAT | 3113-3072 | Reverse complement of HSMUT2for |
| HSMUT3for | GGCAAGACGTATACAGAACAGGTTATAGTGAAGTAATTC | 3084-3122 | RgpB with Lys732 → Gln |
| HSMUT3rev | GAATTACTTCACTATAACCTGTTCTGTATACGTCTTGCC | 3122-3084 | Reverse complement of HSMUT3for |
| S679T684for | CTGTAGCTGTACAAGGTAAGACGATACAAGTAGAAAGTCCTGCTG | 2932-2976 | RgpB with Ser679 → Gln and Thr684 → Gln |
| S679T684rev | CAGCAGGACTTTCTACTTGTATCGTCTTACCTTGTACAGCTACAG | 2976-2932 | Reverse complement of S679T684for |
| T675T682for | GATAAGCCTTATCAAGTAGCTGTACAAGGTAAGCAGATACAAGTAGAAAGTC | 2919-2970 | RgpB with Thr675 → Gln and Thr682 → Gln |
| T675T682rev | GACTTTCTACTTGTATCTGCTTACCTTGTACAGCTACTTGATAAGGCTTATC | 2970-2919 | Reverse complement of T675T682for |
| T724Q-kt-for | GCCGTTCGCATCGCTCAAGAAGGCAAGACGTATAC | 3063-3097 | RgpB with Thr724 → Gln |
| T724Q-kt-rev | GTATACGTCTTGCCTTCTTGAGCGATGCGAACGGC | 3097-3063 | Reverse complement of T724Q-kt-for |
| RgpBD696A-for | GGCTGACGATCTTCGCTATGAACGGCCGTCG | 2980-3010 | RgpB with Asp696 → Ala |
| RgpBD696A-rev | CGACGGCCGTTCATAGCGAAGATCGTCAGCC | 3010-2980 | Reverse complement of RgpBD696A-for |
| RgpBY718F-for | CACAAAACGGCGTGTTTGCCGTTCGCATCGC | 3046-3076 | RgpB with Tyr718 → Phe |
| RgpBY718F-rev | GCGATGCGAACGGCAAACACGCCGTTTTGTG | 3076-3046 | Reverse complement of RgpBY718F-for |
| RgpBY674F-for | CGTAGCCAATGATAAGCCTTTTACTGTAGCTGTATCAGG | 2909-2947 | RgpB with Tyr674 → Phe |
| RgpBY674F-rev | CCTGATACAGCTACAGTAAAAGGCTTATCATTGGCTACG | 2947-2909 | Reverse complement of RgpBY674F-for |
| RgpBT693Qfor | CCTGCTGCCGGGCTGCAAATCTTCGATATG | 2970-2999 | RgpB with Thr693 → Gln |
| RgpBT693Qrev | CATATCGAAGATTTGCAGCCCGGCAGCAGG | 2999-2970 | Reverse complement of RgpBT693Qfor |
| RgpBF695Afor | CCGGGCTGACGATCGCAGATATGAACGGCCG | 2977-3007 | RgpB with Phe695 → Ala |
| RgpBF695Arev | CGGCCGTTCATATCTGCGATCGTCAGCCCGG | 3007-2977 | Reverse complement of RgpBF695Afor |
| RgpBN698Afor | CGATCTTCGATATGGCCGGCCGTCGTGTAGC | 2986-3016 | RgpB with Asn698 → Ala |
| RgpBN698Arev | GCTACACGACGGCCGGCCATATCGAAGATCG | 3016-2986 | Reverse complement of RgpBN698Afor |
a
All the primers were purchased from GeneWorks, Pty. Ltd., Australia.
b
Primer alignment location in rgpB of P. gingivalis W50 according to the rgpB sequence of GenBank accession number AF007124. nt, nucleotides.