Team:SDU-Denmark/project-t

From 2010.igem.org

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== Retinal Generator ==
== Retinal Generator ==
====Retinal requirements of light-sensing proteins====
====Retinal requirements of light-sensing proteins====
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Proteorhodopsins and other related light-sensing proteins such as the rhodopsins found in human retinal pigment often require a supply of retinal to function. In fact retinal is the molecule responsible for the initial activation, as it undergoes photoizomerization after being struck by a photon. It is this change in conformation of the retinal molecule that is relayed through the entire rhodopsin-transducer complex to activate/deactivate the CheW/A complex in the cytosol. Thus either an external supply of retinal or an internal supply of genes coding for enzymes in the retinal biosynthesis pathway are required, if we wish to se phototactic behaviour in our cells.<br><br>
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Proteorhodopsins and other related light-sensing proteins such as the rhodopsins found in human retinal pigment often require a supply of retinal to function. In fact retinal is the molecule responsible for the initial activation, as it undergoes photoizomerization after being struck by a photon. It is this change in conformation of the retinal molecule that is relayed through the entire rhodopsin-transducer complex to activate/deactivate the CheW/A complex in the cytosol. Thus either an external supply of retinal or an internal supply, generated by means of genes coding for enzymes in the retinal biosynthesis pathway are required, if we wish to see phototactic behaviour in our cells.<br><br>
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Many plants and microbes have complete retinal biosynthesis pathways integrated into their genomes, to help drive their rhodopsins. In these organisms rhodopsins play an essential role, not only in photosensation but also directly in energy production, in fact in some organisms rhodopsins are used to create proton motive force directly by pumping protons out into the extracellular space using light energy to drive the process. Humans and other animals on the other hand often only have enzymes coding for the final steps of the pathway, more on which later. They rely on a supply of retinal precursors or Vitamin A (a group consisting of retinal and it's metabolites) in their diet. This is why Vitamin A defficiency causes night-blindness as an early symptom in humans.<br><br>
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Many plants and microbes have complete retinal biosynthesis pathways in their genomes, to help drive their rhodopsins. In these organisms rhodopsins play an essential role, not only for photosensing but also directly in energy production. In fact in some organisms rhodopsins are used to create proton motive force directly by pumping protons out into the extracellular space using light energy to drive the process. Humans and other animals on the other hand often only have enzymes coding for the final steps of the pathway, more on which later. They rely on a supply of retinal precursors or vitamin A (a group of molecules consisting of retinal and it's metabolites) in their diet. This is why vitamin A deficiency causes night-blindness as an early symptom in humans.<br><br>
==== Retinal biosynthesis ====
==== Retinal biosynthesis ====
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Retinal is synthesised from the enzymatic cleavage of some carotenes. In our system we focus on cleavage of beta-carotene, partly because it yields 2 all-trans retinal molecules which is the molecule we desire, and partly because the beta-carotene biosynthesis pathway has been introduced to ''E. coli'' already by the Cambridge 2009 iGEM team.
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Retinal is also synthesized from the enzymatic cleavage of some carotenes. In our system we focus on cleavage of beta-carotene, partly because it yields 2 all-trans retinal molecules which are the molecules we desire, and partly because the beta-carotene biosynthesis pathway has already been introduced to ''E. coli'' by the Cambridge 2009 iGEM team.
The Cambridge construct uses genes from the plant pathogen ''Pantoea ananatis'' and our construct completes the pathway to retinal with a gene from the common fruit fly, ''Drosophila melanogaster''.  <br></p>
The Cambridge construct uses genes from the plant pathogen ''Pantoea ananatis'' and our construct completes the pathway to retinal with a gene from the common fruit fly, ''Drosophila melanogaster''.  <br></p>
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The Cambridge 2009 construct consists of four genes ''crtE'', ''crtB'', ''crtI'' and ''crtY'' from ''P. ananatis'' that together make up the pathway that converts farnesyl pyrophosphate to beta-carotene, which is a precursor for retinal. farnesyl pyrophosphate is naturally pressent in E. coli. <br>
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The Cambridge 2009 construct consists of four genes ''crtE'', ''crtB'', ''crtI'' and ''crtY'' from ''P. ananatis'' that together make up the pathway that converts farnesyl pyrophosphate to beta-carotene, which is a precursor for retinal. farnesyl pyrophosphate is naturally pressent in ‘’E. coli’’. <br>
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• ''crtE'' encodes the protein geranyl-geranyl pyrophosphate synthase that converts farnesyl pyrophosphate to geranyl-geranyl pyrophosphate by elongating it by one unit of isopentenyl. <br>
• ''crtE'' encodes the protein geranyl-geranyl pyrophosphate synthase that converts farnesyl pyrophosphate to geranyl-geranyl pyrophosphate by elongating it by one unit of isopentenyl. <br>
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To introduce the final step from beta-carotene to retinal, we use the gene ''ninaB'' from ''D. melanogaster''. This gene encodes the protein beta-carotene 15,15’-monooxygenase, which cleaves beta-carotene to produce two molecules of retinal under the consumption of oxygen. <br>
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To introduce the final step from beta-carotene to retinal, we use the gene ''ninaB'' from ''D. melanogaster''. This gene encodes the protein beta-carotene 15,15’-monooxygenase, which cleaves beta-carotene to produce two molecules of trans-retinal under the consumption of oxygen. <br>
We have inserted the part K343006 into a different plasmid from the K274210 part since both parts are very long, so a plasmid containing both wouldn't have been viable.
We have inserted the part K343006 into a different plasmid from the K274210 part since both parts are very long, so a plasmid containing both wouldn't have been viable.

Revision as of 17:08, 26 October 2010