Part:BBa_K2263003

α1G calcium ion channel

α1G, which is also known as CACNA1G or Cav3.1, is the α1G subunit of the calcium voltage-gated channel. In our system, it is functional as a depolarising channel which allows the electrophysiological oscillations to occur. Furthermore, its functionality as a calcium channel allowed us to visualise a rhythm using calcium imaging in one of our earliest experiments.

In our project, we used α1G to generate a stable electrophysiological oscillation in HEK cells. (Read more about that on our wiki: http://2017.igem.org/Team:KU_Leuven ) In this project, we used this gene encoded by the wildtype sequence, and not the biobrick-compatible version we present here. However, we have made a great effort to introduce the silent mutations in a smart and codon-optimising way, and we hope other teams will use and characterise this part in the future.

Even though we were excited to deliver an α1G biobrick to the depository, we have unfortunately been unable to adapt and clone this ion channel to follow the biobrick standards. We suspect these troubles are largely the result of the large size of this gene, as it is almost 7000 nucleotides in length. We suggest that future teams interested in using this gene order it as several gBlock fragments and clone them together using Gibson assembly or restriction cloning.

For teams that want to use this biobrick, we suggest to clone the part into a mammalian expression vector, followed by an internal ribosome entry site (IRES) and a fluorescent marker. In this case, the part and the marker are transcribed at the same time, but translated independently. In such a system, the marker indicates that the part is transcribed in the cell, but they are not physically linked. This is beneficial, as a linked marker protein may affect or hinder the protein function.

IRES sequences and fluorescent markers can be found throughout the registry. Furthermore, many labs using mammalian expression vectors will own empty vectors containing an IRES and a marker. You could clone this part directly into such a vector.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1258
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 4955
  Illegal BamHI site found at 344
  Illegal BamHI site found at 2118
  Illegal BamHI site found at 6777
  Illegal BamHI site found at 6879
  Illegal XhoI site found at 5174
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 89
  Illegal NgoMIV site found at 2544
  Illegal NgoMIV site found at 5363
  Illegal NgoMIV site found at 6515
  Illegal NgoMIV site found at 6653
  Illegal NgoMIV site found at 6759
  Illegal AgeI site found at 4382
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 1359
  Illegal BsaI site found at 4098
  Illegal BsaI.rc site found at 1198
  Illegal BsaI.rc site found at 5321
  Illegal BsaI.rc site found at 6385
  Illegal BsaI.rc site found at 6434
  Illegal SapI site found at 2619
  Illegal SapI site found at 4299