TABLE 2.

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

Mutation	Mutagenic oligonucleotidea	Indigo formationb
F352G	5′-GTTCAGCGAACGGGCGGGCCTGCTGG-3′	+
F352A	5′-GTTCAGCGAACGGCCGGGCCTGCTGG-3′	+
F352T	5′-GTTCAGCGAACGACCGGGCCTGCTGG-3′	++
F352I	5′-GTTCAGCGAACGATCGGGCCTGCTG-3′	++
F352Lc	5′-TTCAGCGAACGCTCGGGCCTGC-3′	++
F352Vc	5′-TTCAGCGAACGGTCGGGCCTGC-3′	+
F352W	5′-GTTCAGCGAACGTGGGGGCCTGCTGG-3′	−
F352Y	5′-TTCAGCGAACGTACGGGCCTGCTGG-3′	−

^aUnderlined bases indicate the position of the eliminated restriction site, AclI. Base changes are in bold. 

^bIndigo formation was monitored after 8 h as described in Materials and Methods. ++, colonies dark blue; +, colonies light blue; −, no blue color [corresponds to negative control, JM109(DE3)(pT7-5)]. 

^cThe construction of the F352L and F352V mutations was previously reported (38).