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Team:SydneyUni Australia/Project/Results
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| - | <h2> | + | <h2>ToMO degrades DCA</h2> |
<ul> | <ul> | ||
| - | <li> | + | <li>Early in our project we needed to find a suitable monooxygenase to begin degradation of DCA by one of the two degradation pathways. <br><br><b>MISSING PICTURES </b></li> |
| - | <li> | + | <li>Toluene-o-xylene monooxygenase (ToMO) from Pseuodomonas stutzeri OX1 has been shown to oxidise xylenes, toluene, benzene, styrene, napthalene (Bertoni et al, 1996) as well as tetrachloroethene, trichloroethene, dichloroethene and vinyl chloride (Shim et al, 2001). The enzyme was optimised for chlorinated ethene degradation (Varder & Wood, 2005), and gifted to our host lab in the plasmid pBS(Kan)ToMO.</li> |
| - | <li> | + | <ul> |
| + | <li>Bertoni, G., Bolognese, F., Galli, E., & Barbieri, P. (1996). Cloning of the genes for and characterization of the early stages of toluene and o-xylene catabolism in Pseudomonas stutzeri OX1. Applied and environmental microbiology, 62(10), 3704-3711.</li> | ||
| + | <li>Shim, H., Ryoo, D., Barbieri, P., & Wood, T. (2001). Aerobic degradation of mixtures of tetrachloroethylene, trichloroethylene, dichloroethylenes, and vinyl chloride by toluene-o-xylene monooxygenase of Pseudomonas stutzeri OX1.Applied microbiology and biotechnology, 56(1-2), 265-269.</li> | ||
| + | <li>Vardar, G., & Wood, T. K. (2005). Protein engineering of toluene-o-xylene monooxygenase from Pseudomonas stutzeri OX1 for enhanced chlorinated ethene degradation and o-xylene oxidation. Applied microbiology and biotechnology, 68(4), 510-517.</li> | ||
| + | </ul> | ||
| + | <li>We showed that ToMO can begin degradation of DCA through an assay for chloride ions (link to protocol) released as DCA is converted to chloroacetaldehyde. To our knowledge this has’t been shown by anyone else before. </li> | ||
| + | <li>Table of results from Cl- assay showing how awesome ToMO is:<br><br><img src="https://static.igem.org/mediawiki/2013/f/fe/SydneyUni2013_Results_TomoDCA.png"></li> | ||
| + | <li>This is pretty cool, but during the middle of the year we decided to try synthesising the whole pathway rather than building it by conventional cloning. The length of the ToMO gene cluster meant it was too expensive for us to continue working with it.</li> | ||
| + | <li>E. coli expressing ToMO converts indol to an indo-coloured compound:<br><br><img src="https://static.igem.org/mediawiki/2013/9/97/SydneyUni2013_Results_TomoIndigo.jpg"></li> | ||
</ul> | </ul> | ||
<h2>Info</h2> | <h2>Info</h2> | ||
Revision as of 10:18, 26 September 2013
Project Results
ToMO degrades DCA
- Early in our project we needed to find a suitable monooxygenase to begin degradation of DCA by one of the two degradation pathways.
MISSING PICTURES - Toluene-o-xylene monooxygenase (ToMO) from Pseuodomonas stutzeri OX1 has been shown to oxidise xylenes, toluene, benzene, styrene, napthalene (Bertoni et al, 1996) as well as tetrachloroethene, trichloroethene, dichloroethene and vinyl chloride (Shim et al, 2001). The enzyme was optimised for chlorinated ethene degradation (Varder & Wood, 2005), and gifted to our host lab in the plasmid pBS(Kan)ToMO.
- Bertoni, G., Bolognese, F., Galli, E., & Barbieri, P. (1996). Cloning of the genes for and characterization of the early stages of toluene and o-xylene catabolism in Pseudomonas stutzeri OX1. Applied and environmental microbiology, 62(10), 3704-3711.
- Shim, H., Ryoo, D., Barbieri, P., & Wood, T. (2001). Aerobic degradation of mixtures of tetrachloroethylene, trichloroethylene, dichloroethylenes, and vinyl chloride by toluene-o-xylene monooxygenase of Pseudomonas stutzeri OX1.Applied microbiology and biotechnology, 56(1-2), 265-269.
- Vardar, G., & Wood, T. K. (2005). Protein engineering of toluene-o-xylene monooxygenase from Pseudomonas stutzeri OX1 for enhanced chlorinated ethene degradation and o-xylene oxidation. Applied microbiology and biotechnology, 68(4), 510-517.
- We showed that ToMO can begin degradation of DCA through an assay for chloride ions (link to protocol) released as DCA is converted to chloroacetaldehyde. To our knowledge this has’t been shown by anyone else before.
- Table of results from Cl- assay showing how awesome ToMO is:
- This is pretty cool, but during the middle of the year we decided to try synthesising the whole pathway rather than building it by conventional cloning. The length of the ToMO gene cluster meant it was too expensive for us to continue working with it.
- E. coli expressing ToMO converts indol to an indo-coloured compound:
Info
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