Team:SDU-Denmark/project-t

From 2010.igem.org

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====Bacterial flagellar motility====
====Bacterial flagellar motility====
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Bacteria have evolved many modes of propulsion for the microscale environments they inhabit. At these scales materials behave very different than at macroscale, and of particular interest to us is the way liquids seem more viscous [[http://2010.igem.org/Team:SDU-Denmark/project-t#References 1]]. One of the many ways bacteria move around in liquids, is by means of flagella. A single flagellum is a thin filament around 100-150 Å thick, that extends many cell lengths out from the cell [[http://2010.igem.org/Team:SDU-Denmark/project-t#References 2]]. It consists mainly of flagellin subunits that assemble into a helical structure forming a long hollow cylindrical filament [[http://2010.igem.org/Team:SDU-Denmark/project-t#References 3]]. In ''E. coli'' the mean number of flagella per cell is 4 [[http://2010.igem.org/Team:SDU-Denmark/project-t#References 4]], but there is a wide variance between strains, and even between individual cells of each strain. The environment around the cell also has a large influence on how many flagellae are present, or if they are present at all [[http://2010.igem.org/Team:SDU-Denmark/project-t#References 4]]. Flagella rotate to generate force that allows bacterial cells to swim through fluids in characteristic patterns, more on which later, in search of better conditions for proliferation or survival [[http://2010.igem.org/Team:SDU-Denmark/project-t#References 5]]. Normally flagellated strains of ''E. coli'' can achieve speeds up to 20µm/sec [[http://2010.igem.org/Team:SDU-Denmark/project-t#References 4]], and considering a cell length of only 1-2µm, this is an impressive feat indeed.<br><br>
Bacteria have evolved many modes of propulsion for the microscale environments they inhabit. At these scales materials behave very different than at macroscale, and of particular interest to us is the way liquids seem more viscous [[http://2010.igem.org/Team:SDU-Denmark/project-t#References 1]]. One of the many ways bacteria move around in liquids, is by means of flagella. A single flagellum is a thin filament around 100-150 Å thick, that extends many cell lengths out from the cell [[http://2010.igem.org/Team:SDU-Denmark/project-t#References 2]]. It consists mainly of flagellin subunits that assemble into a helical structure forming a long hollow cylindrical filament [[http://2010.igem.org/Team:SDU-Denmark/project-t#References 3]]. In ''E. coli'' the mean number of flagella per cell is 4 [[http://2010.igem.org/Team:SDU-Denmark/project-t#References 4]], but there is a wide variance between strains, and even between individual cells of each strain. The environment around the cell also has a large influence on how many flagellae are present, or if they are present at all [[http://2010.igem.org/Team:SDU-Denmark/project-t#References 4]]. Flagella rotate to generate force that allows bacterial cells to swim through fluids in characteristic patterns, more on which later, in search of better conditions for proliferation or survival [[http://2010.igem.org/Team:SDU-Denmark/project-t#References 5]]. Normally flagellated strains of ''E. coli'' can achieve speeds up to 20µm/sec [[http://2010.igem.org/Team:SDU-Denmark/project-t#References 4]], and considering a cell length of only 1-2µm, this is an impressive feat indeed.<br><br>
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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 [http://partsregistry.org/Part:BBa_K274210 Cambridge 2009 ] iGEM team.
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 [http://partsregistry.org/Part:BBa_K274210 Cambridge 2009 ] iGEM team.
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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 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>
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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>
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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• ''crtY'' encodes the protein lycopene B-cyclase and converts lycopene to beta-carotene. <br><br>
• ''crtY'' encodes the protein lycopene B-cyclase and converts lycopene to beta-carotene. <br><br>
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The pathway (including the step that generates retinal) is summed up below: <br><br></p>
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The pathway (including the step that generates retinal) is summed up below: <br><br>
[[image:http://2010.igem.org/wiki/images/c/cb/Team-SDU-Denmark-Retinal_generator.png |500px|thumb|Figure 3: The retinal Biosynthesis pathway.]]
[[image:http://2010.igem.org/wiki/images/c/cb/Team-SDU-Denmark-Retinal_generator.png |500px|thumb|Figure 3: The retinal Biosynthesis pathway.]]
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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>
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.
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And now for all of the truly brainy stuff!
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Revision as of 00:30, 27 October 2010