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Team:SDU-Denmark/project-t
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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 start 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 start 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 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 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]. | ||
| - | <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 it's own, by transferring the gen for the enzyme that cleaves beta-carotene to retinal from flies (drosophilia). 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.<br><br> | + | <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 it's own, by transferring the gen for the enzyme that cleaves beta-carotene to retinal from flies (drosophilia). 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:<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 it's 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 it's signalling function can be coupled to other pathways than chemotaxis.<br><br> | ||
'''Biobrick design:''' | '''Biobrick design:''' | ||
Revision as of 12:37, 3 July 2010
Theory
In this section we will review the theory behind our approach to establishing a flow through a microtube.
Phototaxis
Background:
We want to be able to control, switching on and off, our flow through a remote signal. Our preferred signal is light, since light does not have any effect on the rest of the system and only interacts with the membrane receptor in E.Coli. This means that the probability of unwanted side effects is minimized, since there are no excess interactions between the signal and its target environment.
The type of light that we will use for this is bluelight, which functions as a repellent in our case. This will make the bacteria want to get away from the light source which in turn results in an increased tumbling frequency, why will be explained a little further down this text. Since we chose E.Coli as our model organism and wanted to use a light signal, we would have to increase it's sensitivity to bluelight, which naturally is very, very small. Thorugh research we found out that teh Halobacterium Salinarum has a very well researched phototaxis mechanism, where the individual membrain domais role in thr process had been solved AND transferred to E.Coli. Which means that we would have to pick up on that research and create this mechanism as biobricks.
The following model shows the way we want to couple the phototaxis pathway to E.Coli's natural chemotaxis pathway. This is almost identical to the phototaxis pathway in Halobacteria except that the HtrII is directly coupled to CheA, so that there is no Tsr involved.
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 start 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 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].
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 it's own, by transferring the gen for the enzyme that cleaves beta-carotene to retinal from flies (drosophilia). For the accumulation of beta-carotene we will use the biobrick BBa_K274210, which was constructed by the Cambridge team in 2009 [1]. 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:
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 it's signalling function can be coupled to other pathways than chemotaxis.
Biobrick design:
Retinal generator biobrick: BBa_K343002 (Sandboxed)
SopII-HtrII-Tsr fusion,chimer coding sequence: BBa_K343003 (Sandboxed)
Hyperflagellation
To maximize the microflow system's effectivity, we want to increase the force each single bacterium can generate. Since the main motor for the flow is the flagellum we will have to modify this factor for increasing the force. Flagella in E.Coli rotate at a maximum speed around 6000 rpm
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