Talk:20.109(S13):Design mutant (Day1): Difference between revisions
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| Blue | | Blue | ||
|M72S | |M72S | ||
|CCT GAA TTC CTT ACT AGC ATG | |CCT GAA TTC CTT ACT AGC ATG GCT AGA AAA ATG AAG GAC AC | ||
|Phe F | |Phe F | ||
|EcoRI | |EcoRI |
Revision as of 14:09, 12 March 2013
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 | |||||
Yellow | |||||
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 | D80 | 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 | |||||
Orange | |||||
Yellow | |||||
Green | |||||
Blue | |||||
Pink | |||||
Purple | |||||
Platinum |