Talk:20.109(S13):Design mutant (Day1)

Reference mutant choicesl

The mutants below were chosen based on promising student results in previous years, then tested by the teaching faculty.

Mutation X#Z	Primer (forward)	Silent mutation	New restriction site	Known effect
D24H	C GAC AAG GAT GGG CAC GGT ACC ATC ACC -ANS	Gly25	KpnI	Affinity decrease and cooperativity increase
E67K	GGC AAT GGT ACG ATT TAC TTT CCT AAA TTT CTT ACT ATG ATG GC	Gly63	RsaI	Small affinity increase, and perhaps slight cooperativity decrease
T79P	GA AAA ATG AAG GAC CCA GAC AGC GAA GAG GAA ATC C	Thr79	PpuMI	Small affinity decrease and perhaps slight cooperativity increase
M124S	GAT GAA GAA GTT GAT GAA AGT ATA CGG GAA GCA GAT ATC GAT GG	Arg126	AccI	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.

T/R

Please list all amino acid residues with respect to calmodulin itself rather than the chimeric protein inverse pericam.

Team colour	Mutation X#Z	Primer (forward)	Silent mutation	New restriction site	Reference mutant choice
Teaching example	S101L	GG AAC GGC TAC ATC CTC GCT GCG CAG TTA CGT CAC G	Ala 103	FspI	N/A
Red	G24Q	C GAC AAG GAT GGG GAC CAG ACC ATCACC ACC AAG GAA CTT G	Thr 28	Sty1
Orange
Yellow
Green	M71D				T79P
Blue
Pink
Purple
Platinum

W/F

Please list all amino acid residues with respect to calmodulin itself rather than the chimeric protein inverse pericam.

Team colour	Mutation X#Z	Primer (forward)	Silent mutation	New restriction site	Reference mutant choice
Teaching example	S101L	GG AAC GGC TAC ATC CTC GCT GCG CAG TTA CGT CAC G	Ala 103	FspI	N/A
Red
Orange
Yellow
Green
Blue
Pink
Purple
Platinum