Mutant choices for positive control
The mutants below were chosen based on promising student results in previous years, then tested by the teaching faculty.
||New restriction site
||C GAC AAG GAT GGG CAC GGT ACC ATC ACC -ANS
||Affinity decrease and cooperativity increase (both substantial)
||GGC AAT GGT ACG ATT TAC TTT CCT AAA TTT CTT ACT ATG ATG GC
||Small affinity increase
||GA AAA ATG AAG GAC CCA GAC AGC GAA GAG GAA ATC C
||Small affinity decrease and perhaps slight cooperativity increase
||GAT GAA GAA GTT GAT GAA AGT ATA CGG GAA GCA GAT ATC GAT GG
||Affinity decrease and cooperativity increase
Note that we also investigated D22H and D129P, but both of these turned out to destroy calcium response, and in low salt conditions to eliminate fluorescence itself. The limited choices above aren't meant to imply that you must narrowly investigate these residues or ones just like them.