Difference between revisions of "Part:BBa K1943021"
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<partinfo>BBa_K1943021 short</partinfo>
 
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Transient receptor potential channel 5 (TRPC5) is a calcium ion permeable channel that can be activated by mechanic force.[1] In our study, audiogenetics is defined as a method of gene regulation by applying sound precisely and safely and the external sound signal is transduced through TRPC5. In order to get mutated TRPC5 with high sensitivity to mechanical stress, we need to find out the specific fragment of TRPC5 which is related to stress sensing. According to the structure of TRPC5, we finally chose the ankyrin repeats region as the mutated region.[2] We used GeneMorph II Random Mutagenesis Kit (Provided by Agilent Technologies Company) to make a library of mutated ankyrin repeats by error-prone PCR. We submitted 5 TRPC5 from our library and we used site-directed mutation to mutate EcoRI and PstI restriction sites in the coding sequence based on codon degeneracy.
  
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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.)
 
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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.
 
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The mutated amino acid sites are as listed:
 
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{| class="wikitable"
 
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! Part        
 
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! Original amino acids
 
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! Amino acid after mutation
 
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|-
 
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| BBa_K1943022
 
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| Trs(240)+Ala(27)
 
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| Glu(240)+Thr(27)
 
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|-
 
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| BBa_K1943023
 
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| Ala(27)
 
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| Thr(27)
 
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|-
 
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| BBa_K1943024
 
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| Met(134)+Ala(27)
 
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| Ile(134)+Thr(27)
 
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|-
 
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| BBa_K1943025
 
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| Glu(236)+Ala(27)
 
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| Lys(236)+Thr(27)
 
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|-
 
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| BBa_K1943026
 
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| Gln(128)+Ala(27)
 
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| Arg(128)+Thr(27)
 
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|}
 
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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.
 
  
 
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Revision as of 02:13, 22 October 2016


TRPC5 Transient receptor potential channel 5 (TRPC5) is a calcium ion permeable channel that can be activated by mechanic force.[1] In our study, audiogenetics is defined as a method of gene regulation by applying sound precisely and safely and the external sound signal is transduced through TRPC5. In order to get mutated TRPC5 with high sensitivity to mechanical stress, we need to find out the specific fragment of TRPC5 which is related to stress sensing. According to the structure of TRPC5, we finally chose the ankyrin repeats region as the mutated region.[2] We used GeneMorph II Random Mutagenesis Kit (Provided by Agilent Technologies Company) to make a library of mutated ankyrin repeats by error-prone PCR. We submitted 5 TRPC5 from our library and we used site-directed mutation to mutate EcoRI and PstI restriction sites in the coding sequence based on codon degeneracy.


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