Part:BBa_K838002

Melanopsin

Melanopsin is a photosensitive protein membrane bound G protein coupled receptor. Photoactivation of a cis retinal (R) chromo- phore changes the comformation of melanopsin which in turn activates G alpha type G protein. The G alpha sets off a signaling cascade which eventually results in the activation of transient receptor potential channels which will allow calcium influx. The increased calcium concentration in the cell activates the calmodulin -> calcineurin -> NFAT pathway. The end result is the activation of genes situated next to NFAT promoter elements.

Just to recapitulate, this part is a membrane bound receptor. All other pathways used are endogenous.

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Usage and Biology

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 282
  Illegal PstI site found at 853
  Illegal PstI site found at 1311
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 282
  Illegal PstI site found at 853
  Illegal PstI site found at 1311
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1369
  Illegal BamHI site found at 45
  Illegal BamHI site found at 541
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 282
  Illegal PstI site found at 853
  Illegal PstI site found at 1311
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 282
  Illegal PstI site found at 853
  Illegal PstI site found at 1311
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 711
  

How to use

1) Obtain the parts: To implement a light activated gene switch using this protein, all one needs is this part and any gene to express in a mammalian cell.

2) Cloning:

First clone the part into a mammalian expression vector such as [http://products.invitrogen.com/ivgn/product/V79020?ICID=search-product pcDNA3.1(+)] or [http://products.invitrogen.com/ivgn/product/V04450 pCEP4]. These are the two main expression vectors we used. To use the melanopsin pathway, you also need to clone any gene you want to express into the mammalian vector [http://www.promega.com/resources/protocols/product-information-sheets/a/pgl430-vector-protocol/ pGL4.30].

3) Transfection Co-transfect the combination of photoreceptive protein and readout construct of your choice in mammalian cells!