Team:Lethbridge/Project/Catechol Degradation
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Catechol is a toxic organic molecule commonly found in tailings ponds. Furthermore, many other toxic compounds, such as naphthenic acids, can be metabolized into catechol. <i>Pseudomonas putida</i> demonstrates great metabolic diversity and is able to utilize a wide range of carbon sources, including molecules few other organisms can break down<sup>3</sup>. The <html><a href="https://2008.igem.org/Team:University_of_Lethbridge" target="new"><font color="green"> Lethbridge 2008 iGEM</font></a></html> isolated the <i>XylE</i> gene (<html><a href="http://partsregistry.org/Part:BBa_K147002" target="new"><font color="green">BBa_K147002</font></a></html>) from <i>P.putida</i>, which codes for catechol 2,3-dioxygenase (XylE). However, the <html><a href="https://2008.igem.org/Team:Edinburgh" target="new"><font color="green">Edinburgh 2008 iGEM</font></a></html> team isolated the <i>XylE</i> gene (<html><a href="http://partsregistry.org/Part:BBa_K118021" target="new"><font color="green">BBa_K118021</font></a></html>) from <i>P.putida</i>, along with a ribosomal binding site (rbs), so we chose to work with this part. This year we have engineered <i>Escherichia coli</i> DH5αto express XylE. XylE rapidly converts catechol into 2-hydroxymuconic semialdehyde (2-HMS). 2-HMS is a non-toxic, bright yellow molecule that can be metalbolized by <i>E.coli</i> DH5α. | Catechol is a toxic organic molecule commonly found in tailings ponds. Furthermore, many other toxic compounds, such as naphthenic acids, can be metabolized into catechol. <i>Pseudomonas putida</i> demonstrates great metabolic diversity and is able to utilize a wide range of carbon sources, including molecules few other organisms can break down<sup>3</sup>. The <html><a href="https://2008.igem.org/Team:University_of_Lethbridge" target="new"><font color="green"> Lethbridge 2008 iGEM</font></a></html> isolated the <i>XylE</i> gene (<html><a href="http://partsregistry.org/Part:BBa_K147002" target="new"><font color="green">BBa_K147002</font></a></html>) from <i>P.putida</i>, which codes for catechol 2,3-dioxygenase (XylE). However, the <html><a href="https://2008.igem.org/Team:Edinburgh" target="new"><font color="green">Edinburgh 2008 iGEM</font></a></html> team isolated the <i>XylE</i> gene (<html><a href="http://partsregistry.org/Part:BBa_K118021" target="new"><font color="green">BBa_K118021</font></a></html>) from <i>P.putida</i>, along with a ribosomal binding site (rbs), so we chose to work with this part. This year we have engineered <i>Escherichia coli</i> DH5αto express XylE. XylE rapidly converts catechol into 2-hydroxymuconic semialdehyde (2-HMS). 2-HMS is a non-toxic, bright yellow molecule that can be metalbolized by <i>E.coli</i> DH5α. | ||
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We will then use our <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="green">lumazine synthase (LS) microcompartment</font></a></html> to isolate the XylE protein. The LS has been previously characterized and shown that it is able to encapsulate other molecules. We can target a protein into the LS through selectively mutating five of the interior amino acids of the LS to glutamate. By attaching a positively charged arginine tag to the C-terminus of the protein for targeting you can selectively target the tagged protein into the compartment (<html><a href="https://2009.igem.org/Team:Lethbridge/Modeling" target="new"><font color="green">Lethbridge 2009 Modeling</font></a></html>) (Seebeck et al., 2006). We will then purify the complex and use for application in tailings ponds water. Once we have demonstrated that we are successfully able to isolate the XylE we will then add more enzymes for the bioremediation of the tailings ponds. | We will then use our <html><a href="https://2010.igem.org/Team:Lethbridge/Project/Compartamentalization"><font color="green">lumazine synthase (LS) microcompartment</font></a></html> to isolate the XylE protein. The LS has been previously characterized and shown that it is able to encapsulate other molecules. We can target a protein into the LS through selectively mutating five of the interior amino acids of the LS to glutamate. By attaching a positively charged arginine tag to the C-terminus of the protein for targeting you can selectively target the tagged protein into the compartment (<html><a href="https://2009.igem.org/Team:Lethbridge/Modeling" target="new"><font color="green">Lethbridge 2009 Modeling</font></a></html>) (Seebeck et al., 2006). We will then purify the complex and use for application in tailings ponds water. Once we have demonstrated that we are successfully able to isolate the XylE we will then add more enzymes for the bioremediation of the tailings ponds. | ||
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===<font color="white">References=== | ===<font color="white">References=== | ||
- | 1) Government of Alberta. "<html><a href="http://www.oilsands.alberta.ca/resource.html" target=""><font color="green"> About the Resource</font></a></html>." Alberta's Oil Sands. 2010. Government of Alberta. 25 October 2010. | + | 1) Government of Alberta. "<html><a href="http://www.oilsands.alberta.ca/resource.html" target=""><font color="green">About the Resource</font></a></html>." Alberta's Oil Sands. 2010. Government of Alberta. 25 October 2010. |
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2) Davies, M. P.; Rice, S. (16–19 January 2001). "An alternative to conventional tailing management - "dry stack" filtered tailings". <i>Proceedings of the Eighth International Conference on Tailings and Mine Waste</i>. Fort Collins, Colorado, US: Balkema. pp. 411–422. | 2) Davies, M. P.; Rice, S. (16–19 January 2001). "An alternative to conventional tailing management - "dry stack" filtered tailings". <i>Proceedings of the Eighth International Conference on Tailings and Mine Waste</i>. Fort Collins, Colorado, US: Balkema. pp. 411–422. | ||
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3) Newton C.M. Gomes, Irina A. Kosheleva, Wolf-Rainer Abraham, Kornelia Smalla (2005) Effects of the inoculant strain <i>Pseudomonas putida</i> KT2442 (pNF142) and of naphthalene contamination on the soil bacterial community. FEMS Microbiology Ecology 54 (1), 21–33. | 3) Newton C.M. Gomes, Irina A. Kosheleva, Wolf-Rainer Abraham, Kornelia Smalla (2005) Effects of the inoculant strain <i>Pseudomonas putida</i> KT2442 (pNF142) and of naphthalene contamination on the soil bacterial community. FEMS Microbiology Ecology 54 (1), 21–33. | ||
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