Talk:20.109(S13):Design mutant (Day1): Difference between revisions
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Line 60: | Line 60: | ||
|- | |- | ||
| Red | | Red | ||
|G24Q | |G24Q<br>''Note: actually G25Q'' | ||
|C GAC AAG GAT GGG GAC CAG ACC ATCACC ACC AAG GAA CTT G | |C GAC AAG GAT GGG GAC CAG ACC ATCACC ACC AAG GAA CTT G | ||
|Thr 28 | |Thr 28 | ||
Line 67: | Line 67: | ||
|- | |- | ||
| Orange | | Orange | ||
|E140R, E141R | |E140R, E141R<br>''Note: actually E139-140R'' | ||
|CGATGGCGATGGCCAAGTAAACTATCGACGGTTTGTACAAAATG | |CGATGGCGATGGCCAAGTAAACTATCGACGGTTTGTACAAAATG<br>''Note: extra 'A,' ordered CGATGGCGATGGCCAAGTAAACTATCGACGGTTTGTACAAATG'' | ||
|Gly 133 | |Gly 133 | ||
|BtgZI | |BtgZI | ||
Line 81: | Line 81: | ||
|- | |- | ||
| Green | | Green | ||
| | |M71R | ||
|CCT GAA TTC CTT ACT AGG ATG GCT AGA AAA ATG AAG GAC ACA G | |CCT GAA TTC CTT ACT AGG ATG GCT AGA AAA ATG AAG GAC ACA G | ||
|Phe 68 | |Phe 68 | ||
Line 88: | Line 88: | ||
|- | |- | ||
| Blue | | Blue | ||
|M72S | |M72S<br>''Note: actually M71S'' | ||
|CCT GAA TTC CTT ACT AGC ATG GCT AGA AAA ATG AAG GAC AC | |CCT GAA TTC CTT ACT AGC ATG GCT AGA AAA ATG AAG GAC AC | ||
|Phe F | |Phe F | ||
Line 139: | Line 139: | ||
|- | |- | ||
| Red | | Red | ||
| | |G23E | ||
| | |CTTCTCATTATTCGACAAGGATGAGGACGGTACCATCACCAC | ||
| | |Gly25 | ||
| | |Kpn1 | ||
| | |E67K | ||
|- | |- | ||
| Orange | | Orange | ||
Line 153: | Line 153: | ||
|- | |- | ||
| Yellow | | Yellow | ||
|M59T | |M59T<br>''Note: actually M51T'' | ||
|CA GAA TTG CAG GAT ACG ATC AAT GAA GTC GAC GCT GAT GGC | |CA GAA TTG CAG GAT ACG ATC AAT GAA GTC GAC GCT GAT GGC | ||
|Asp63 | |Asp63 | ||
Line 175: | Line 175: | ||
| Pink | | Pink | ||
|G25S | |G25S | ||
|CGACAAGGATGGGGACAGCACCATCACCACAAAGGAAC | |CGACAAGGATGGGGACAGCACCATCACCACAAAGGAAC<br>''Note: missing silent mutation, ordered CGACAAGGATGGGGACAGCACCATCACCACCAAGGAAC'' | ||
|Thr29 | |Thr29 | ||
|Sty1 | |Sty1 | ||
Line 181: | Line 181: | ||
|- | |- | ||
| Purple | | Purple | ||
| | |A128E | ||
| | |GAA ATG ATA AGG GAA GAA GAT ATC GAT GGC GAT GGC C | ||
| | |Gly 132 | ||
| | |BtgZI | ||
| | |M124S | ||
|- | |- | ||
| Platinum | | Platinum | ||
|R125P | |R125P<br>''Note: actually R126P'' | ||
|GTTGATGAAATGATACCGGAAGCAGATATCGATGGCGATGGCC | |GTTGATGAAATGATACCGGAAGCAGATATCGATGGCGATGGCC | ||
|Gly 131 | |Gly 131 |
Latest revision as of 14:09, 20 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 Note: actually G25Q |
C GAC AAG GAT GGG GAC CAG ACC ATCACC ACC AAG GAA CTT G | Thr 28 | Sty1 | D24H |
Orange | E140R, E141R Note: actually E139-140R |
CGATGGCGATGGCCAAGTAAACTATCGACGGTTTGTACAAAATG Note: extra 'A,' ordered CGATGGCGATGGCCAAGTAAACTATCGACGGTTTGTACAAATG |
Gly 133 | BtgZI | E67K |
Yellow | K77E | CTATGATGGCTAGAAAAATGGAGGACACAGACTCC | Ser81 | BsaXI | T79P |
Green | M71R | CCT GAA TTC CTT ACT AGG ATG GCT AGA AAA ATG AAG GAC ACA G | Phe 68 | EcoRI | T79P |
Blue | M72S Note: actually M71S |
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 | Gly25 | Kpn1 | E67K |
Orange | F16S | GCA GAG TTC AAA GAA GCC TCC TCA TTA TTC GAC AAG GAT GG | None | BseR1 | M124S |
Yellow | M59T Note: actually M51T |
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 Note: missing silent mutation, ordered CGACAAGGATGGGGACAGCACCATCACCACCAAGGAAC |
Thr29 | Sty1 | D24H |
Purple | A128E | GAA ATG ATA AGG GAA GAA GAT ATC GAT GGC GAT GGC C | Gly 132 | BtgZI | M124S |
Platinum | R125P Note: actually R126P |
GTTGATGAAATGATACCGGAAGCAGATATCGATGGCGATGGCC | Gly 131 | BtgZI | E67K |