TABLE 2.
Primer sequences used for mutagenesis
| Gene | Desired mutationa | Primerb | Primer sequence (5′→3′)c |
|---|---|---|---|
| racE1 | Cys77Ala | racE1C77AFor | 5′-GGCTCTAGTTGTAGCAGCGAATACTGCTGCAGCTGC-3′ |
| racE1C77ARev | 5′-GCAGCTGCAGCAGTATTCGCTGCTACAACTAGAGCC-3′ | ||
| racE1 | Cys188Ala | racE1C188AFor | 5′-GATACGTTAATTCTTGGGGCGACGCATTATCCACTTTTAGAG-3′ |
| racE1C188ARev | 5′-CTCTAAAAGTGGATAATGCGTCGCCCCAAGAATTAACGTATC-3′ | ||
| racE2 | Cys74Ala | racE2C74AFor | 5′-CAAAATGTTAGTTATTGCAGCGAATACAGCAACTGCAGTTGTATTAG-3′ |
| racE2C74ARev | 5′-CTAATACAACTGCAGTTGCTGTATTCGCTGCAATAACTAACATTTTG-3′ | ||
| racE2 | Cys185Ala | racE2C185AFor | 5′-GATACACTTATTTTAGGTGCGACACATTATCCGATTTTAGGTCC-3′ |
| racE2C185ARev | 5′-GGACCTAAAATCGGATAATGTGTCGCACCTAAAATAAGTGTATC-3′ |
a
Residues were selected for conservative mutagenesis due to their close proximity to d-glutamate in the homology models generated from the B. subtilis RacE crystal structure (43).
b
Primers were designed in accordance with the QuikChange site-directed mutagenesis protocol by Stratagene (La Jolla, CA) and were synthesized by Integrated DNA Technologies (Coralville, IA).
c
Underlined sequences indicate engineered codon mutations.