Difference between revisions of "Part:BBa K902048:Experience"

Latest revision as of 03:09, 4 October 2012

XylE (J33204) was biobricked under the tetR promoter (R0040). This construct was confirmed by sequencing and used in visual assays. The visual assay shown below demonstrated that the XylE gene was expressed with the tetR promoter and that the resulting enzyme (catechol 2,3-dioxygenase) was functional. The colour change to bright yellow indicated the conversion of catechol to 2-hydroxymuconic semialdehyde.

Fig.1 Cells transformed with the part BBa_K902048 were grown overnight in BHI media. The cells were spun down and washed in M9 minimal media. The tube on the left contains the supernatant without catechol. The tube on the right was brought to a catechol concentration of 0.1 M.

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 in bacteria, 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 [http://2012.igem.org/Team:Calgary/Notebook/Protocols/decatecholization 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 as shown in Figure 4. While the identity of this compound is currently unknown, there are changes occuring to some of the catechol breakdown products.

Figure 6: Gas chromatograph of catechol degradation assay using Jeotgalicoccus sp. ATCC 8456 a species of bacteria that converts fatty acids into alkenes. This identified a similar peak change in the PetroBrick with a retention time of 10.5 min as shown in Figure 4. This provides additional support that the PetroBrick and this organism can further degrade catechol into breakdown products.

Figure 7: Mass spectra of i>Jeotgalicoccus</i> sp. ATCC 8456/xylE co-culture retention peak at 10.5 min as shown in Figure 6. This peak is similar to the peak from the Petrobrick/xylE co-culture, suggesting the breakdown product for both of these cultures is modified catechol from xylE. The identification of this compound is ongoing.

Based on our GC-MS results, we were able to show the appearance of a new peak when cells expressing xylE and the PetroBrick were co-cultured. Although we don't know the exact identity of this peak, it is distinct form our control. Interestingly, a similar peak appeared when cells expressing our xylE construct were co-cultured with Jeotgalicoccus sp. ATCC 8456 cells. This suggests that although we don't know the exact identity of this new peak, it is likely that it may be in fact a further breakdown product of catechol. This is a very promising result, as it suggests that in addition to converting naphthenic acids into hydrocarbons, we may also be able to break down catechol, one of the other major toxic components in tailings ponds.

Applications of BBa_K902048

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−	~~</html>~~[[image:UCalgary2012_Catechol_assay_%282%29.jpg\|500px\|thumb\|Fig.1 Cells transformed with the part BBa_K902048 were grown overnight in BHI media. The cells were spun down and washed in M9 minimal media. The tube on the left contains the supernatant without catechol. The tube on the right was brought to a catechol concentration of 0.1 M.\|center]]	+	[[image:UCalgary2012_Catechol_assay_%282%29.jpg\|500px\|thumb\|Fig.1 Cells transformed with the part BBa_K902048 were grown overnight in BHI media. The cells were spun down and washed in M9 minimal media. The tube on the left contains the supernatant without catechol. The tube on the right was brought to a catechol concentration of 0.1 M.\|center]]

	After verifying that we could in fact degrade catechol into 2-hydroxymuconate semialdehyde using our <i>xylE</i> construct ([https://parts.igem.org/Part:BBa_J33204 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 in bacteria, this could be particularly useful.		After verifying that we could in fact degrade catechol into 2-hydroxymuconate semialdehyde using our <i>xylE</i> construct ([https://parts.igem.org/Part:BBa_J33204 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 in bacteria, this could be particularly useful.