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	D24H
Orange	E140R, E141R	CGATGGCGATGGCCAAGTAAACTATCGACGGTTTGTACAAAATG	Gly 133	BtgZI	E67K
Yellow	K77E	CTATGATGGCTAGAAAAATGGAGGACACAGACTCC	Ser81	BsaXI	T79P
Green	M71D	CCT GAA TTC CTT ACT AGG ATG GCT AGA AAA ATG AAG GAC ACA G	Phe 68	EcoRI	T79P
Blue	M72S	CCT GAA TTC CTT ACT AGC ATG GCT AGA AAA ATG AAG GAC AC	Phe F	EcoRI	M124S
Pink
Purple	M144R	GAG GAG TTT GTA CAA AGG ATG ACA GCA AAG TAA GAA TTC GAA GC	Glu139	BseRI	M124S
Platinum	D80R	CT AGA AAA ATG AAG GAC ACA CGT AGC GAA GAG GAA ATC CGA GAA GC	Asp80	AflIII	M124S

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	G23E	CTTCTCATTATTCGACAAGGATGAGGACGGTACCATCACCAC		Kpn1
Orange	F16S	GCA GAG TTC AAA GAA GCC TCC TCA TTA TTC GAC AAG GAT GG	None	BseR1	M124S
Yellow	M59T	CA GAA TTG CAG GAT ACG ATC AAT GAA GTC GAC GCT GAT GGC	Asp63	AccI	M124S
Green	M76G	CTATGATGGCTAGAAAAGGGAAGGACACAGACAGCGAGGAGG	Glu 81	BseRI	T79P
Blue	D22E	TTC TCA TTA TTC GAC AAG GAA GGG GAC GGT ACC ATC ACC	Gly 25	Kpn1	D24H
Pink	G25S	CGACAAGGATGGGGACAGCACCATCACCACAAAGGAAC	Thr29	Sty1	D24H
Purple	A128E	GAA ATG ATA AGG GAA GAA GAT ATC GAT GGC GAT GGC C	Gly 410	BtgZI	M124S
Platinum	R125P	GTTGATGAAATGATACCGGAAGCAGATATCGATGGCGATGGCC	Gly 131	BtgZI	E67K