Team:SDU-Denmark/project-t

From 2010.igem.org

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(Retinal biosynthesis)
(Photosensor)
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The way the halobacterial pathway works is that the photonreceptor is a protein called sensory rhodopsin II, which absorbs the blue light and in response changes it's conformation. HtrII is just a transducer and signals this to CheA, which in turn gets phosphorylated and afterwards passes the phosphate group on to CheB. Phosphorylated CheB binds to the flagellar motor switch, so that the flagella starts rotating clockwise, which induces the tumbling motility pattern. The more CheY gets phosphorylated the higher the tumbling frequency will be.  
The way the halobacterial pathway works is that the photonreceptor is a protein called sensory rhodopsin II, which absorbs the blue light and in response changes it's conformation. HtrII is just a transducer and signals this to CheA, which in turn gets phosphorylated and afterwards passes the phosphate group on to CheB. Phosphorylated CheB binds to the flagellar motor switch, so that the flagella starts rotating clockwise, which induces the tumbling motility pattern. The more CheY gets phosphorylated the higher the tumbling frequency will be.  
Our focus is to get this working in ''E. coli'', which craves a few extra steps. First we will have to link the SopII and HtrII domains together with a 9 amino acid residue linker, so that the signal transducing happens succesfully in ''E. coli''. We also have to fuse HtrII and Tsr in their cytoplasmic domains, which is the HAMP domain, that both proteins contain. Fusion in this HAMP domain effectively couples the phototaxic receptors to the chemotaxis pathway, so that a phototactic effect is possible in ''E. coli''. These construction informations were obtained from the article: ''An Archaeal Photosignal-Transducing Module Mediates Phototaxis in Escherichia coli'' by Spudich et Al. [http://jb.asm.org/cgi/content/full/183/21/6365]. That this system is functional in vitro in ''E. coli'' has also been shown by Spudich et Al in the article ''Photostimulation of a Sensory Rhodopsin II/HtrII/Tsr Fusion Chimera Activates CheA-Autophosphorylation and CheY-Phosphotransfer in Vitro† '' [http://www.ncbi.nlm.nih.gov/pubmed/14636056].
Our focus is to get this working in ''E. coli'', which craves a few extra steps. First we will have to link the SopII and HtrII domains together with a 9 amino acid residue linker, so that the signal transducing happens succesfully in ''E. coli''. We also have to fuse HtrII and Tsr in their cytoplasmic domains, which is the HAMP domain, that both proteins contain. Fusion in this HAMP domain effectively couples the phototaxic receptors to the chemotaxis pathway, so that a phototactic effect is possible in ''E. coli''. These construction informations were obtained from the article: ''An Archaeal Photosignal-Transducing Module Mediates Phototaxis in Escherichia coli'' by Spudich et Al. [http://jb.asm.org/cgi/content/full/183/21/6365]. That this system is functional in vitro in ''E. coli'' has also been shown by Spudich et Al in the article ''Photostimulation of a Sensory Rhodopsin II/HtrII/Tsr Fusion Chimera Activates CheA-Autophosphorylation and CheY-Phosphotransfer in Vitro† '' [http://www.ncbi.nlm.nih.gov/pubmed/14636056].
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<br> We will only have to add retinal to the system, which is needed for proper function of the fusion,chimera-protein. Therefore we want ''E. coli'' to produce retinal on its own, by transferring the gene for the enzyme that cleaves beta-carotene to retinal from flies (drosophila melanogaster). For the accumulation of beta-carotene we will use the biobrick BBa_K274210, which was constructed by the Cambridge team in 2009 [https://2009.igem.org/Team:Cambridge]. We will expand this brick's functionality by coupling it with the enzyme that cleaves beta-carotene to retinal. In that way we will be able to construct a retinal generator with the help of Cambridge's and our part. Here is a model of the retinal generator:<br><br><html><img width="600px" height="400px" src="https://static.igem.org/mediawiki/2010/c/cb/Team-SDU-Denmark-Retinal_generator.png"></img></html><br><br>  
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<br> We will only have to add retinal to the system, which is needed for proper function of the fusion,chimera-protein. Therefore we want ''E. coli'' to produce retinal on its own, by transferring the gene for the enzyme that cleaves beta-carotene to retinal from flies (drosophila melanogaster). For the accumulation of beta-carotene we will use the biobrick K274210, which was constructed by the Cambridge team in 2009 [https://2009.igem.org/Team:Cambridge]. We will expand this brick's functionality by coupling it with the enzyme that cleaves beta-carotene to retinal. In that way we will be able to construct a retinal generator with the help of Cambridge's and our part. Here is a model of the retinal generator:<br><br><html><img width="600px" height="400px" src="https://static.igem.org/mediawiki/2010/c/cb/Team-SDU-Denmark-Retinal_generator.png"></img></html><br><br>  
In the end we want to split the whole fusion, chimer into two biobricks that can be fused as a composite part. By doing this we hopefully introduce biobricks that give ''E. coli'' phototaxic abilities and also introduce modularity into the complex, so that its signalling function can be coupled to other pathways than chemotaxis.<br><br>
In the end we want to split the whole fusion, chimer into two biobricks that can be fused as a composite part. By doing this we hopefully introduce biobricks that give ''E. coli'' phototaxic abilities and also introduce modularity into the complex, so that its signalling function can be coupled to other pathways than chemotaxis.<br><br>
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Revision as of 18:05, 24 October 2010