Part:BBa_K1943021

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.

[1] ↑ Zholos AV., TRPC5., Handb Exp Pharmacol., Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/24756705.

[2] ↑ Beck, A., et al., Conserved gating elements in TRPC4 and TRPC5 channels., J Biol Chem., Retrieved from https://www.ncbi.nlm.nih.gov/pubmed/23677990.

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