Team:Calgary/Project/OSCAR/CatecholDegradation

From 2012.igem.org

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<p>After verifying that we could in fact degrade catechol into 2-hydroxymuconate semialdehyde using our <i>xylE</i>  construct (<a href=http://partsregistry.org/Part:BBa_J33204>BBa_J33204</a>), we wondered if we could take this any further.  What if we could convert this by-product page into hydrocarbons too?  As catechol is the breakdown product of a number of different degradation pathways, this could be particularly useful.</p>
<p>After verifying that we could in fact degrade catechol into 2-hydroxymuconate semialdehyde using our <i>xylE</i>  construct (<a href=http://partsregistry.org/Part:BBa_J33204>BBa_J33204</a>), we wondered if we could take this any further.  What if we could convert this by-product page into hydrocarbons too?  As catechol is the breakdown product of a number of different degradation pathways, this could be particularly useful.</p>
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<p>As 2-hydroxymuconate semialdehyde can be further metabolized to pyruvate and acetaldehyde (Harayama S et al., 1987), it seemed possible that these products could be routed into the fatty acid biosynthesis pathway and converted to alkanes using the PetroBrick or the OleT enzyme.  Given that the Catechol 2,3-dioxygenase reaction is extracellular, it creates a possible scenario in which cells with the <i>xylE</i> construct could be co-cultured with Petrobrick-containing cells to cooperatively metabolise catechol into hydrocarbons. </p>
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<p>As 2-hydroxymuconate semialdehyde can be further metabolized to pyruvate and acetaldehyde (Harayama S et al., 1987), it seemed possible that these products could be routed into the fatty acid biosynthesis pathway and converted to alkanes using the PetroBrick or the <i>OleT</i> enzyme.  Given that the Catechol 2,3-dioxygenase reaction is extracellular, it creates a possible scenario in which cells with the <i>xylE</i> construct could be co-cultured with Petrobrick-containing cells to cooperatively metabolise catechol into hydrocarbons. </p>
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<p> In order to test this, we followed this <a href=http://2012.igem.org/Team:Calgary/Notebook/Protocols/decatecholization>protocol</a>, where we co-cultured cells expressing our <i>xylE</i> construct with either <i>E. coli</i> cells expressing the PetroBrick, or <i>Jeotgalicoccus sp. ATCC 8456</i> cells expressing OleT.  in the presence of catechol.
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<p> In order to test this, we followed this <a href=http://2012.igem.org/Team:Calgary/Notebook/Protocols/decatecholization>protocol</a>, where we co-cultured cells expressing our <i>xylE</i> construct with either <i>E. coli</i> cells expressing the PetroBrick, or <i>Jeotgalicoccus sp. ATCC 8456</i> cells expressing <i>OleT</i>.  in the presence of catechol.

Revision as of 01:55, 4 October 2012

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Catechol Degradation

Catechol is a toxic compound found in tailings ponds that is a by-product of polyaromatic hydrocarbon metabolism (Vaillancourt et al., 2006, Schweigert et al., 2001)). The chemical properties of catechol allow it to react with biomolecules, causing cellular damage including DNA damage, enzyme inactivation and membrane uncoupling (Schweigert et al., 2001).

Catechol is characterized as having a benzene ring with two hydroxyl groups at the 2,3 position. It can be converted to 2-hydroxymuconic acid by the enzyme catechol 2,3-dioxygenase, encoded by the xylE gene on the Tol plasmid of Pseudomonas putida (Nakai et al., 1983).

Currently the registry has two BioBricks available of xylE. One contained xylE with its native ribosome-binding site (BBa_J33204), while the other part contained xylE under the glucose-repressible promoter cstA (BBa_K118021). Given that E. coli is grown in the presence of glucose, we designed a new construct to keep xylE repressed by using the tetR promoter (BBa_R0040).

Figure 1: BioBrick genetic circuit for catechol degradation showing xylE under the tetR promoter

Catechol 2,3-dioxygenase is an extradiol dioxygenase which cleaves catechol adjacent to the two hydroxyl groups. When this occurs 2-hydroxymuconate semialdehyde is produced, which is yellow in colour. This change in colour allows for visual assay to assess the activity of XylE.

Figure 2: Catechol 2,3-dioxygenase (XylE) converts catechol to 2-Hydroxymuconate semialdehyde in the presence of oxygen. Adapted from Shu et al., 1995.

The visual assays were performed with E. coli cells transformed with (BBa_K118021) as well as with E. coli cells transformed with the newly constructed part (BBa_K902048) by bringing the supernatant of an overnight culture to a concentration of 0.1 M of catechol. When the part (BBa_K118021) was used, the pellet was first washed in M9-MM and centrifuged before catechol was added to the supernatant. This was necessary to avoid the glucose in the LB from repressing the cstA promoter (BBa_K118011). 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 was indicative that catechol was breaking down into 2-Hydroxymuconate semialdehyde, which was exactly what we expected! This assay was completed by following the protocol written by the 2008 Edinburgh iGEM team.

Figure 3: Results of the catechol visual assay using xylE BBa_K118021. Cultures were grown overnight in LB and the pellets were washed with M9-MM at various times (From left to right: 0 min, 5 min, 10 min, 15 min, and 20 min.). Cells were then spun down and catechol was added to the supernatant to 0.1 M. The amount of time didn't affect the colour change in the cultures containing the XylE gene. The right most tube was a culture of E. coli cells without the xylE gene that was used as a control. The controls supernatant remained clear when the catechol was added.

Converting Catechol into hydrocarbons?

After verifying that we could in fact degrade catechol into 2-hydroxymuconate semialdehyde using our xylE construct (BBa_J33204), we wondered if we could take this any further. What if we could convert this by-product page into hydrocarbons too? As catechol is the breakdown product of a number of different degradation pathways, this could be particularly useful.

As 2-hydroxymuconate semialdehyde can be further metabolized to pyruvate and acetaldehyde (Harayama S et al., 1987), it seemed possible that these products could be routed into the fatty acid biosynthesis pathway and converted to alkanes using the PetroBrick or the OleT enzyme. Given that the Catechol 2,3-dioxygenase reaction is extracellular, it creates a possible scenario in which cells with the xylE construct could be co-cultured with Petrobrick-containing cells to cooperatively metabolise catechol into hydrocarbons.

In order to test this, we followed this protocol, where we co-cultured cells expressing our xylE construct with either E. coli cells expressing the PetroBrick, or Jeotgalicoccus sp. ATCC 8456 cells expressing OleT. in the presence of catechol.

Figure 4: Gas chromatograph of catechol degradation assay using the PetroBrick. While there is limited differences between xylE incubated with and without the Petrobrick, there was one peak with a retention time of 10.5 min which was dramatically increased in the co-culture.
Figure 5: Mass spectra of the Petrobrick/xylE co-culture retention peak at 10.5 min from Figure 3. While the identity of this compound is unknown to us presently, it is clear that there is changes occuring to some of the catechol breakdown products.
Figure 6: Gas chromatograph of catechol degradation assay using Micrococcus cadicans a species of bacteria shown to be able to convert fatty acids into alkenes. This identified a similar peak change is in the Petrobrick with a retention time of 10.5 min. This provides additional support for the idea that the Petrobrick and this organism can further degrade catechol breakdown products.
Figure 7: Mass spectra of Micrococcus/xylE co-culture retention peak at 10.5 min from Figure 5. The similarity of this peak to the one found from the PetroBrick co-culture suggests that there may be similar breakdown products for both of these enzymes suggesting that we are capable of modifying catechol further than with xylE alone. We have yet to identify what this compound is.