Difference between revisions of "Part:BBa K838002"
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__NOTOC__ | __NOTOC__ | ||
<partinfo>BBa_K838002 short</partinfo> | <partinfo>BBa_K838002 short</partinfo> | ||
| − | Melanopsin is a photosensitive protein | + | [[Image:Fussenegger-scheme.jpg|thumb| [http://www.sciencemag.org/content/332/6037/1565.abstract Fussenegger et al.]]] |
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| + | 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. | ||
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| + | Just to recapitulate, this part is a membrane bound receptor. All other pathways used are endogenous. | ||
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===Usage and Biology=== | ===Usage and Biology=== | ||
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| + | 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. | ||
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| + | ===How do I implement this switch in my mammalian cells?=== | ||
| + | |||
| + | '''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:''' | ||
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| + | 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]. | ||
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| + | [[Image:Team-EPF-Lausanne_pNFAT.png|500px]] | ||
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| + | '''3) Transfection''' | ||
| + | Co-transfect the combination of photoreceptive protein and readout construct of your choice in mammalian cells! | ||
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| + | |||
| + | [[Image:Team_EPF_Lausanne_complexswitch.png|400px]] | ||
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| + | '''4) 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. | ||
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| + | '''5) Test the expression''' | ||
| + | It is possible to perform a Western blot using an [http://www.biocompare.com/9776-Antibodies/589319-Rabbit-AntiMouse-Melanopsin-Antibody-Saporin-Conjugated/ anti mouse melanopsin antibody]. To look at the functionality of melanopsin, calcium imaging is the most straight forward technique, because it allows to compare calcium influx between melanopsin transfected cells and non transfected cells. | ||
| + | Look at the Experience page and our [http://2012.igem.org/Team:EPF-Lausanne wiki] for more infos! | ||
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
<partinfo>BBa_K838002 SequenceAndFeatures</partinfo> | <partinfo>BBa_K838002 SequenceAndFeatures</partinfo> | ||
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===Functional Parameters=== | ===Functional Parameters=== | ||
<partinfo>BBa_K838002 parameters</partinfo> | <partinfo>BBa_K838002 parameters</partinfo> | ||
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Latest revision as of 06:45, 6 October 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
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 do I implement this switch in my mammalian cells?
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!
4) 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.
5) Test the expression
It is possible to perform a Western blot using an [http://www.biocompare.com/9776-Antibodies/589319-Rabbit-AntiMouse-Melanopsin-Antibody-Saporin-Conjugated/ anti mouse melanopsin antibody]. To look at the functionality of melanopsin, calcium imaging is the most straight forward technique, because it allows to compare calcium influx between melanopsin transfected cells and non transfected cells.
Look at the Experience page and our [http://2012.igem.org/Team:EPF-Lausanne wiki] for more infos!
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 282
Illegal PstI site found at 853
Illegal PstI site found at 1311 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 282
Illegal PstI site found at 853
Illegal PstI site found at 1311 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1369
Illegal BamHI site found at 45
Illegal BamHI site found at 541 - 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 282
Illegal PstI site found at 853
Illegal PstI site found at 1311 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 282
Illegal PstI site found at 853
Illegal PstI site found at 1311 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 711