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.

Usage and Biology

This switch uses an endogenous pathway in a cell to activate the transcription of a gene of interest in a cell. Melanopsin is embedded in the membrane and activates this pathway upon activation by blue light. The key protein is NFAT, imported into the cell nucleus as a consequence of melanopsin activation, which causes it to bind PNFAT, a NFAT responsive promoter placed upstream the gene of interest.

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!

3) Illumination 10 seconds pulses every 10 seconds with blue light (468 nm) is recommended for the melanopsin protein. To build a set up like the one we used, take a look at our [http://2012.igem.org/Team:EPF-Lausanne/Bioreactor Bioreactor assembly page], where we explain what components you need, how to assemble them and how to program your illumination device.

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