TABLE 2.
Primers used in this study
| Primer name | Descriptiona | Sequence (5′ to 3′) |
|---|---|---|
| Btoxoid-RN2-fw | Forward primer for β-toxoid cloning in pNZ8048 | CGTTGCCATGGAGAAAAAATTTATTTCATTAGTTATAG |
| Btoxoid-RN2-rv | Reverse primer for β-toxoid cloning in pNZ8048 | CGCTCTAGATTAAATAGCTGTTACTTTGTGAG |
| Btox-fw_BstEII | Forward primer for Btoxoid_SS, introduces BstEII and NdeI sites | TCGGTGACCCATATGAAGAAAAAATTTATTTCATTAG |
| Btoxoid-RN2-XhoI | Reverse primer for β-toxoid, introduces XhoI site | CGCCTCGAGTTAAATAGCTGTTACTTTGTGAG |
| RNlacZ-fw | Universal primer on pUC18-derived MCS | GTGAGCGGATAACAATTTCACACAGG |
| RNlacZ-rv | Universal primer on pUC18-derived MCS | GGTTTTCCCAGTCACGACGTTGTAA |
| PhtrA_fw-KpnI | Forward primer for promotor region htrA, introduces KpnI site | CGTGAGGTACCGGCTTCTGTTTCTGCC |
| PhtrA_rv | Reverse primer for promotor region htrA, introduces HindIII site | CATCACGAAGCTTATCCATCATGTTCACTCCG |
| PykoJ-fw1 | Forward primer for amplification of ykoJ promoter, introduces KpnI site | CTGGTACCGCAGTGAATCCATCTGCCATGAC |
| PykoJ-rv1 | Reverse primer for amplification of ykoJ promoter, introduces HindIII site | ACTAAGCTTGAGCATTTGTGAGCCCTCCTTTGT |
| ykoJ-fw1 | Forward primer to amplify ykoJ for overexpression, introduces BstEII site | TGTGGTGACCCAAATGCTCAAGAAAAAATGGATGGTCGGTCTTTTAG |
| ykoJ-rv1 | Reverse primer to amplify ykoJ for overexpression, introduces XhoI site | ACACTCGAGTTAGTCATCTATCTCCTGTTTGATAATG |
| up_ykoJ-fw | Forward primer to amplify upstream region of ykoJ, internal pstI site is amplified | CTTTTGCTGCAGCAGCCATTTTAG |
| up_ykoJ-rv | Reverse primer to amplify upstream region of ykoJ, introduces HindIII site | TCTTAAGCTTTTGTGAGCCCTCCTTTGTTT |
| down_ykoJ-fw | Forward primer to amplify downstream region of ykoJ, introduces HindIII site | TCTAAGCTTCAGGAGATAGATGACTAATCAA |
| down_ykoJ-rv | Reverse primer to amplify downstream region of ykoJ, introduces XbaI site | CGATCTAGAAGCATCCAGCTGCATTA |
| btox-change_FW | Universal forward primer for mutagenesis of β-toxin, introduces Eco31I site | CGGTCTCATATTCATCTGAAATGACAACTTTAATAAACTTAAC |
| RV-btox_DAA | Reverse primer, changes DDK → DAA | CGGTCTCAAATAAGCAGCATCGATAAATCTAGCATCTATAGATGCAGTAA |
| RV-btox_ADA | Reverse primer, changes DDK → ADA | CGGTCTCAAATAAGCATCAGCGATAAATCTAGCATCTATAGATGCAGTAA |
| RV-btox_AAK | Reverse primer, changes DDK → AAK | CGGTCTCAAATATTTAGCAGCGATAAATCTAGCATCTATAGATGCAGTAA |
| RV-btox_DDA | Reverse primer, changes DDK → DDA | CGGTCTCAAATAAGCATCATCGATAAATCTAGCATCTATAGATGCAGTAA |
| RV-btox_DAK | Reverse primer, changes DDK → DAK | CGGTCTCAAATATTTAGCATCGATAAATCTAGCATCTATAGATGCAGTAA |
| RV-btox_ADK | Reverse primer, changes DDK → ADK | CGGTCTCAAATATTTATCAGCGATAAATCTAGCATCTATAGATGCAGTAA |
a
MCS, multiple cloning site.