Reporter

Part:BBa_K1943023

Designed by: Shixin Lu, Shiqiang Tang   Group: iGEM16_SUSTech_Shenzhen   (2016-10-13)


TRPC5-12

Transient receptor potential channel 5 (TRPC5) is a mechanosensitive (MS) channel which takes charge of hearing in our project. Future teams could use original TRPC5 plasmid (BBa_K1943021) to sense sound or other kinds of mechanical forces. Furthermore, we randomly mutated the ankyrin region of TRPC5 channel by PCR. We expected the ankyrin region to be a crucial part for sense mechanical forces based on the structure of TRPC5.(Detailed methods are shown in our notebook.)

These mutated TRPC5 plasmids(BBa_K1943022, BBa_K1943023, BBa_K1943024, BBa_K1943025, BBa_K1943026) have all been sequenced. Because of limited time, we have not yet transfected them into cells to observe whether the channels are more sensitive to sound and mechanical force or not. Future teams are welcomed to use and finish testing them.

The mutated amino acid sites are as listed:

Part Original amino acids Amino acid after mutation
BBa_K1943022 Trs(240)+Ala(27) Glu(240)+Thr(27)
BBa_K1943023 Ala(27) Thr(27)
BBa_K1943024 Met(134)+Ala(27) Ile(134)+Thr(27)
BBa_K1943025 Glu(236)+Ala(27) Lys(236)+Thr(27)
BBa_K1943026 Gln(128)+Ala(27) Arg(128)+Thr(27)

TRPC5 sequence contains 1 EcoRⅠ RE site and 2 PstⅠ RE sites, which are unacceptable for BioBricks. Therefore, we did site-mutation by PCR for three times. Detailed information are shown in our notebook.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 930
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 167
    Illegal BglII site found at 1520
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2539
    Illegal BsaI.rc site found at 1627
    Illegal BsaI.rc site found at 2054
    Illegal SapI site found at 1722


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