Talk:20.109(S12):Start-up protein engineering (Day1)

Mutant choices for positive control

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
E67K	GGC AAT GGT ACG ATT TAC TTT CCT AAA TTT CTT ACT ATG ATG GC	Gly63	RsaI	Small affinity increase
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.

TR lab

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	Positive control choice
Teaching example	S101L	GG AAC GGC TAC ATC CTC GCT GCG CAG TTA CGT CAC G	Ala 103	FspI	N/A
Red	V136T	GCA GAT ATC GAT GGT GAT GGC CAA ACA AAC TAT GAA GAG TTT GTA C Note: added two silent mutations, cut one base-pair, and ordered CA GAT ATC GAT GGA GAC GGC CAA ACA AAC TAT GAA GAG TTT GTA C	Gly132/Asp133 -ANS	BsmBI -ANS	M124s
Orange	D24H	G CTG TTC CTA CCC GTG CCA TGG TAG TGG Note: this is the reverse complement of the strand I can most readily understand. Ordered: C GAC AAG GAT GGG CAC GGT ACC ATC ACC -ANS	25Gly	KpnI	T79P
Yellow	D20R	GCCTTCTCATTATTCCGC AAG GAT GGA GAC GGC ACC ATC ACC	25Gly (23Gly -ANS)	BSMBI	E67K
Green	E67R	GGC AAT GGA ACG ATT TAC TTT CCT CGA TTT CTT ACT ATG ATG GCT Note: added two silent mutations, ordered GGC AAT GGT ACC ATT TAC TTT CCT CGA TTT CTT ACT ATG ATG GCT	Gly61/Thr62 -ANS	Acc65I -ANS	E67K
Blue	D57N	G ATC AAT GAA GTC GAT GCT AAT GGC AAT GGA ACG ATT TAC Note: after our discussion, I am ordering G ATC AAT GAA GTC GAT GCT AAT GGC AAT GGT ACC ATT TAC.	Gly61/Thr62	Acc65I	T79P
Pink	M144S	GGC CAA GTA AAC TAT GAA GAG TTT GTA CAA TCG ATG ACA GCA AAG Note: even allowing for >=3 silent mutations, no restriction site addition was possible!	Met 144	N/A	M124S
Purple	F65D	C GAT GCT GAT GGC AAT GGT ACC AAT TAC GAT CCT GAA TTT CTT AC Note: slight error at residue 63. Ordered: C GAT GCT GAT GGC AAT GGT ACC ATT TAC GAT CCT GAA TTT CTT AC	Gly 61, Trp 62	Acc651	T79P

WF lab

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	Positive control choice
Teaching example	S101L	GG AAC GGC TAC ATC CTC GCT GCG CAG TTA CGT CAC G	Ala 103	FspI	N/A
Red	T79F	G GCT AGA AAA ATG AAG GAC TTC GAC AGC GAA GAG GAG ATC CGA G	Glu 84	BseRI	T79P
Orange	V55F	G CAG GAT ATG ATC AAT GAA TTC GAT GCT GAT GGC AAT GGA ACG	N/A	EcoRI	T79P
Yellow	D20K	GAA GCA TTC TCA TTA TTC AAA AAG GAT GGG GAC GGC ACC Note: primer seems to be missing silent mutation. After our discussion, ordered GAA GCA TTC TCA TTA TTC AAA AAG GAT GGG GAC GGC ACC	Ala 15	BsmI	E67K
Green	D96R D95R-ANS	GCA TTT CGT GTT TTT GAC AAG AAT GGG AAC GGC TAC ATC AG	Phe88 Phe89-ANS	Destroys BsmI	T79P
Blue	T26Y	C GAC AAG GAT GGA GAC GGC TAC ATC ACC ACA AAG GAA C Note: primer seems to be missing silent mutation. --> primer now fixed	Gly 23	BsmBI	E67K
Pink	K66E	GGG GAC GGC ACC ATC ACC ACA GAG GAA CTG GGC ACC GTT ATG AGG	Leu 68	XcmI	E67K
Purple	G23W, G25W	(5’) C TCA TTA TTC GAC AAG GAT TGG GAC TGG ACC ATC ACC ACC AAG G	Thr 29	StyI	M124S