Table 2.

Amino acid substitutions in the α subunit of NDO generated by site-directed mutagenesis

MutationMutagenic oligonucleotideaRestriction site changeIndigo formationb
N201A5′-GAGGCACCCGCGGAAGCTTTTGTGGGAGATGCA-3′ HindIII+
N201Q5′-GCACCCGCGGAACAATTTGTGGGAGATGCA-3′ Tsp509I+
N201S5′-CCGCGGAAAGCTTTGTGGGAG-3′ HindIII++
F202L5′-CCGCGGAAAAGCTTGTGGGAGATG-3′ HindIII
F202V5′-CGCGGAAAACGTTGTGGGAGATG-3′ AclI++
V260A5′-ATATTCAGGTGCGCATAGCGCAG-3′ FspI++
V260L5′-GGACGGATATTCAGGGCTCCATAGCGCAGACTTG-3′ BanII++
V260N5′-GACGGATATTCAGGTAACCATAGCGCAGACTTG-3′ BstEII++
W316A5′-GGTGTTTTCAAAGTCGCGAACCCGATCGAC-3′ NruI+++
T351N5′-CTGTTCAGCGAAACTTCGGGCCTGCT-3′RemoveAclI++
T351R5′-CTGTTCAGCGAAGGTTCGGGCCTGCT-3′RemoveAclI+
T351S5′-CTGTTCAGCGAAGCTTCGGGCCTGCT-3′ HindIII+++
F352L5′-TTCAGCGAACGCTCGGGCCTGC-3′RemoveAclI++
F352V5′-TTCAGCGAACGGTCGGGCCTGC-3′RemoveAclI+
W358A5′-GGCCTGCTGGCTTCGCGGAAAGCGACGACA-3′Nonec
D362A5′-GAAAGCGACGCCAATGACAAT-3′ BsaHI
M366W5′-ACGACAATGACAATTG GGAAACAGCTTCGC-3′ MfeI++
  • a Underlined bases indicate the position of the introduced or eliminated restriction site. Base changes are in boldface.

  • b Indigo formation was monitored after 8 h as described in Materials and Methods. +++, colonies were dark blue [corresponds to JM109(DE3)(pDTG141), expressing wild-type NDO]; ++, colonies were medium blue; +, colonies were pale blue; −, no blue color [corresponds to negative control, JM109(DE3)(pT7-5)].

  • c Colonies were pale blue after 12 h.