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Team:Calgary/Project/OSCAR/CatecholDegradation
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| - | <p>Catechol | + | <p><b>Why are we degrading catechol?</b></p> |
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| + | <p>Catechol is a toxic compound found in tailings ponds that is a by-product of polyaromatic hydrocarbon metabolism. Catechol can be degraded by the enzyme catechol 2,3-dioxygenase encoded by the <i>xylE</i> gene on the Tol plasmid of <i>Pseudomonas putida</i> (Nakai <i>et al.</i>, 1983). Previously, there were only two available biobricks of this gene. One contained the <i>xylE</i> with its native RBS site and the other part had an added Pcst promoter, which was glucose-repressible. To tackle to problem of <i>xylE</i> being repressed, when the transformed E.coli was grown in media containing glucose, a TetR promoter was added to the part containing only the gene.</p> | ||
</html>[[File:UCalgary2010_R0040-XylE.png|400px|center]]<html> | </html>[[File:UCalgary2010_R0040-XylE.png|400px|center]]<html> | ||
Revision as of 05:36, 30 September 2012
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Catechol Degradation
Why are we degrading catechol?
Catechol is a toxic compound found in tailings ponds that is a by-product of polyaromatic hydrocarbon metabolism. Catechol can be degraded by the enzyme catechol 2,3-dioxygenase encoded by the xylE gene on the Tol plasmid of Pseudomonas putida (Nakai et al., 1983). Previously, there were only two available biobricks of this gene. One contained the xylE with its native RBS site and the other part had an added Pcst promoter, which was glucose-repressible. To tackle to problem of xylE being repressed, when the transformed E.coli was grown in media containing glucose, a TetR promoter was added to the part containing only the gene.
Catechol 2,3-dioxygenase is an extradiol dioxygenase which cleaves catechol adjacent to the two hydroxyl groups. When this occurs 2-hydroxymuconic semialdehyde is produced, which is yellow in colour. This colour change allows for easy assays to be conducted which show the enzymes activity.
The colour change assays were performed with the newly constructed part by bringing the supernatant of an overnight culture to a concentration of 0.1 M of catechol. The catechol was added to the supernatant because the reaction takes place outside of the cell. Within minutes of the addition of catechol to the supernatant, the solution turned from the pale yellow of LB to a bright yellow. This assay was completed by following the previous assay done by the 2008 Edinburgh iGEM team.
References
Nakai, C., Kagamiyama, H., and Nozaki, M. 1983. Complete nucleotide sequence of the metapyrocatechase gene on the TOL plasmid of Pseudomonas putida. The Journal of Biological Chemistry 258: 2923-2928.
Shu, L., Chiou, Y., Orville, A.M., Miller, M.A., Lipscomb, J.D., and Que, L. 1995. X-ray absorption spectroscopic studies of the Fe(II) active site of catechol 2,3-dioxygenase. Implications for the extradiol cleavage mechanism. Biochemistry 34: 6649-6659.
