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

From OpenWetWare

Jump to: navigation, search

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


Personal tools