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Team:Grenoble-EMSE-LSU/Project/KR
From 2013.igem.org
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| - | <h3>Origin</h3> | + | <h3>Origin</h3><br><br> |
<p>KR was originally engineered from the anm2CP anthomedusa chromoprotein by individual amino acid mutations in order to obtain fluorescence and phototoxicity.[1] | <p>KR was originally engineered from the anm2CP anthomedusa chromoprotein by individual amino acid mutations in order to obtain fluorescence and phototoxicity.[1] | ||
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</li> | </li> | ||
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| + | <li> | ||
| + | <h2>Construction of Plac-RBS-KillerRed</h2><br><br> | ||
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| + | <h3>Introduction</h3> | ||
| + | <p> | ||
| + | This section of the wiki describes the initial construction of a prokaryotic genetic network allowing the expression of KillerRed in <em>E. coli</em><br><br> | ||
| + | The KillerRed gene was in a eukaryotic plasmid when we obtained it initially. To get expression of the protein in <em>E. coli</em>, it is necessary to transfer this gene into a prokaryotic plasmid. The choice of the plasmid and the genetic components inside (Promoter, RBS) is particularly important because we want to control how fast KillerRed kills the cells. To do this we must control the amount of protein in the cells. There is no literature about the effects of KillerRed on cells in low light, so we suspect KillerRed could be toxic even at low doses of light. This represents an additional factor we had to take into account during the construction of the plasmid. | ||
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| + | <h3>Choosing the components</h3><br><br> | ||
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| + | Since the protein could be toxic, we want to control when KillerRed is expressed in the cells, and so we have to choose inducible promoters. In this way, even if cell growth is slowed by KillerRed we can easily measure its effects by plating the culture on agar plates and then counting the number of colonies. But to do that with some precision, and to obtain enough protein for the phototoxic effectwe could make sure that we had a significant cell population in the culture before expressing something potentially toxic. We then had two different inducible promoters to choose from | ||
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| + | </li> | ||
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<li><h2>References</h2> | <li><h2>References</h2> | ||
<p>[1] M.E. Bulina et al., A genetically encoded photosensitizer, <em>Nature Biotechnology</em>, January 2006.<br> | <p>[1] M.E. Bulina et al., A genetically encoded photosensitizer, <em>Nature Biotechnology</em>, January 2006.<br> | ||
[2] Sergei Pletnev et al., Structural Basis for Phototoxicity of the Genetically Encoded Photosensitizer KillerRed, <em>The Journal of Biological Chemistry</em> vol. 284, no. 46, pp. 32028–32039, November 13, 2009.<br> | [2] Sergei Pletnev et al., Structural Basis for Phototoxicity of the Genetically Encoded Photosensitizer KillerRed, <em>The Journal of Biological Chemistry</em> vol. 284, no. 46, pp. 32028–32039, November 13, 2009.<br> | ||
| - | [3] | + | [3] Russell B. Vegh et al., Reactive oxygen species in photochemistry of the red fluorescent protein ‘‘Killer Red’’, <em>Chem. Commun.</em>,2011,47,4887–4889. |
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</p> | </p> | ||
</li> | </li> | ||
Revision as of 12:01, 27 September 2013
