Difference between revisions of "Part:BBa K838002"

Revision as of 19:53, 26 September 2012

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

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] (for pCEP4, cut the backbone with BglII and KpnI; and cut out the expression vector using BamHI and KpnI). 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!

@@ Line 24: / Line 24: @@
 '''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.
+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] (for pCEP4, cut the backbone with BglII and KpnI; and cut out the expression vector using BamHI and KpnI). 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].