Table 2.
Oligonucleotides used in this work
| Target | Primer (forward/reverse, 5′–3′)a |
|---|---|
| Protein expression | |
| lamB | CTAGCTAGCCATGGTTATGCGCGT/CCCAAGCTTTTACCACCAGGCTTCG |
| ail | GGGATCCAGTATTTCTATTGGATATGC/GGTCGACTTAACCCACTTTCACATCATCG |
| ompF | GGGATCCTACGGTAAAGTTGACGC/GGTCGACTTAGGACTGAACATAACCCAG |
| ompA | GGGATCCGGTTTAGTATGGCGTGC/GGTCGACTTAAGCCTGTGGCTGAGTC |
| gp17 | CTAGCTAGCATGGCTAACAAAATTTC/CCCAAGCTTTCAGTTTCGAACTAAG |
| gp17(1–469) | CTAGCTAGCATGGCTAACAAAATTTC/CCCAAGCTTTCAATCGACCATCTTG |
| gp17(1–119) | CTAGCTAGCATGGCTAACAAAATTTC/CCCAAGCTTTCAAGTATCCGCAG |
| gp17(470–569) | CTAGCTAGCGGACTACTCACTACCG/CCCAAGCTTTCAGTTTCGAACTAAG |
| Construction of mutants | |
| Kanamycin resistance cassette | |
| waaA | ATGAATAACCACGGCTCAATGTGGTGCTCCGCCCTCCTAGGTGTAGGCTGGAGCTGCTTC/TTAGTGGCTCCGTTGTGGCAGATAAGGCTCCAACAAATGTCATATGAATATCCTCCTTA |
| lamB | ATGATTACTCTGCGCAAGTTACCAATAGCACTGGCTGTTGGTGTAGGCTGGAGCTGCTTC/TTACCACCAGGCTTCGAATTGCGCACCGAAGGTGACTTCACATATGAATATCCTCCTTA |
| ail | ATGGTTTTTATGAATAAGACATTACTGGTCTCTTCTTTAAGTGTAGGCTGGAGCTGCTTC/TTAGAACCGGTAACCCGCGCCAAGCATCCAAGTACCCACTCATATGAATATCCTCCTTA |
| ompF | ATGAGGGTAATAATAATGATGAAGCGCAATATTCTTGCAGGTGTAGGCTGGAGCTGCTTC/TTAGAACTGGTAAACCAAGCCAACAGCAACAACGTTGTCGCATATGAATATCCTCCTTA |
| ompA | TTGCCTATTTGGATGATAATGAGGCGTAAAATGAAAAAGAGTGTAGGCTGGAGCTGCTTC/TTAAGCCTGTGGCTGAGTCACAACTTCTTTGTAGCCTTTCCATATGAATATCCTCCTTA |
| Chloromycetin resistance cassette | |
| lamB | ATGATTACTCTGCGCAAGTTACCAATAGCACTGGCTGTTGAGATTGCAGCATTACACGTC/TTACCACCAGGCTTCGAATTGCGCACCGAAGGTGACTTCATGTAACGCACTGAGAAGC |
| ail | ATGGTTTTTATGAATAAGACATTACTGGTCTCTTCTTTAAAGATTGCAGCATTACACGTC/TTAGAACCGGTAACCCGCGCCAAGCATCCAAGTACCCACTTGTAACGCACTGAGAAGC |
| ompF | ATGAGGGTAATAATAATGATGAAGCGCAATATTCTTGCAGAGATTGCAGCATTACACGTC/TTAGAACTGGTAAACCAAGCCAACAGCAACAACGTTGTCGTGTAACGCACTGAGAAGC |
| ompA | TTGCCTATTTGGATGATAATGAGGCGTAAAATGAAAAAGAAGATTGCAGCATTACACGTC/TTAAGCCTGTGGCTGAGTCACAACTTCTTTGTAGCCTTTCTGTAACGCACTGAGAAGC |
| Gentamicin resistance cassette | |
| lamB | ATGATTACTCTGCGCAAGTTACCAATAGCACTGGCTGTTGTTAGGTGGCGGTACTTGGG/TTACCACCAGGCTTCGAATTGCGCACCGAAGGTGACTTCACGTCAGGTGGCACTTTTCG |
| ail | ATGGTTTTTATGAATAAGACATTACTGGTCTCTTCTTTAATTAGGTGGCGGTACTTGGG/TTAGAACCGGTAACCCGCGCCAAGCATCCAAGTACCCACTCGTCAGGTGGCACTTTTCG |
| ompF | ATGAGGGTAATAATAATGATGAAGCGCAATATTCTTGCAGTTAGGTGGCGGTACTTGGG/TTAGAACTGGTAAACCAAGCCAACAGCAACAACGTTGTCGCGTCAGGTGGCACTTTTCG |
| Verification of the mutants | |
| waaA | CTGCGGCAGGTTACAAGAAG/ATGTGTGCTAGTCGCAATCC |
| lamB | TACTCAAGAAGGTTACTCG/GTTGGCATAAGTTGCGAAG |
| ail | ACTGGGGAGTGATAGGTTC/TTACTTCTACTCTCTGATTG |
| ompF | CGGCAACGCAACCCGTCTG/CTTCAGAACGGTCATTTTTG |
| ompA | GGGTGGTTTAGTATGGCGTG/TCCTGACCAAAGCGGTAAG |
| Complementation of mutants | |
| lamB | CGCGGATCCGCCCAGAACATC/CCCAAGCTTGTACCAGACAGAGTG |
| ompF | CGCGGATCCCCGTTGCGGCGATGGATG/CCCAAGCTTTTTTCAAACATGTCAGAG |
| ail | CGCGGATCCCCATTGAGCGTGTGAAAC/CCCAAGCTTCAACTTTACCGTCTCAAC |
| ompA | CGCGGATCCTACAGTGTTGTTCTGGG/CCCAAGCTTTACTGAACCCCC |
| waaA | CGCGGATCCGTGCAAAAGCCGTAGTC/CCCAAGCTTCGATTTTAAGCTCTGGG |
a
Underlining indicates endonuclease restriction sites.