Difference between revisions of "Part:BBa K925000"
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===Description=== | ===Description=== | ||
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This part encodes a delta-12 desaturase derived from Synechocystis sp PCC 6803. The enzyme is able to introduce a double bond at the Δ-12 site in the hydrocarbon chain of oleic acid (18:1, Δ9), for its conversion into linoleic acid (18:2 Δ9,12), a polyunsaturated fatty acid (PUFA). Mass spectrometry results show that when feeding oleic acid to E. coli transformed with this part, the bacterial membrane composition is changed, and linoleic acid is observed. | This part encodes a delta-12 desaturase derived from Synechocystis sp PCC 6803. The enzyme is able to introduce a double bond at the Δ-12 site in the hydrocarbon chain of oleic acid (18:1, Δ9), for its conversion into linoleic acid (18:2 Δ9,12), a polyunsaturated fatty acid (PUFA). Mass spectrometry results show that when feeding oleic acid to E. coli transformed with this part, the bacterial membrane composition is changed, and linoleic acid is observed. | ||
===Characterisation=== | ===Characterisation=== | ||
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In order to show desaturase activity of this enzyme, we performed a lipid analysis on Fatty Acid Methyl Esters (FAME) by Gas chromatography–mass spectrometry GC-MS, both on membrane assays and lipid extracts of E. coli expressing this desaturase. | In order to show desaturase activity of this enzyme, we performed a lipid analysis on Fatty Acid Methyl Esters (FAME) by Gas chromatography–mass spectrometry GC-MS, both on membrane assays and lipid extracts of E. coli expressing this desaturase. | ||
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As a control the same FAME-GC analysis was performed in intact cells, and the lipid profiles were compared. Additionally, FAME 18:2 (Δ9,12) standard was run to compare to the unsaturation pattern to that of the 18:2 expected to be observed in the transformed cells. | As a control the same FAME-GC analysis was performed in intact cells, and the lipid profiles were compared. Additionally, FAME 18:2 (Δ9,12) standard was run to compare to the unsaturation pattern to that of the 18:2 expected to be observed in the transformed cells. | ||
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Importantly, the cells were grown in the presence of the substrate 18:1 (Δ9) so that this could be incorporated to the membranes, as this fatty acid is not present in intact E. coli BL21. | Importantly, the cells were grown in the presence of the substrate 18:1 (Δ9) so that this could be incorporated to the membranes, as this fatty acid is not present in intact E. coli BL21. | ||
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[[Image:D16.png]] | [[Image:D16.png]] | ||
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===Results=== | ===Results=== | ||
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Our results indicate that, both in lipid extracts and in our membrane assays derived from cells transformed with Δ-12 desaturase, C18:2 is present, unlike in intact cells. Moreover, this 18:2 has the same unsaturation pattern as our standard, meaning the 18:2 found in the transformed cells is the expected 18:2 (Δ9,12). | Our results indicate that, both in lipid extracts and in our membrane assays derived from cells transformed with Δ-12 desaturase, C18:2 is present, unlike in intact cells. Moreover, this 18:2 has the same unsaturation pattern as our standard, meaning the 18:2 found in the transformed cells is the expected 18:2 (Δ9,12). | ||
===Conclusion=== | ===Conclusion=== | ||
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Lipid profiles of E. coli transformed with our construct show that this Δ-12 desaturase is able to catalyze the desaturation of oleic acid to give linoleic acid. In this way, this BioBrick can be used to build a biosynthetic pathway for PUFAs and Omega-3 fatty acids, along with other desaturases and elongases. | Lipid profiles of E. coli transformed with our construct show that this Δ-12 desaturase is able to catalyze the desaturation of oleic acid to give linoleic acid. In this way, this BioBrick can be used to build a biosynthetic pathway for PUFAs and Omega-3 fatty acids, along with other desaturases and elongases. | ||
Revision as of 21:39, 24 September 2012
Delta-12 desaturase
Short description
Delta-12 desaturase involved in an Omega-3 biosynthetic pathway.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 426
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Description
This part encodes a delta-12 desaturase derived from Synechocystis sp PCC 6803. The enzyme is able to introduce a double bond at the Δ-12 site in the hydrocarbon chain of oleic acid (18:1, Δ9), for its conversion into linoleic acid (18:2 Δ9,12), a polyunsaturated fatty acid (PUFA). Mass spectrometry results show that when feeding oleic acid to E. coli transformed with this part, the bacterial membrane composition is changed, and linoleic acid is observed.
Characterisation
In order to show desaturase activity of this enzyme, we performed a lipid analysis on Fatty Acid Methyl Esters (FAME) by Gas chromatography–mass spectrometry GC-MS, both on membrane assays and lipid extracts of E. coli expressing this desaturase.
As a control the same FAME-GC analysis was performed in intact cells, and the lipid profiles were compared. Additionally, FAME 18:2 (Δ9,12) standard was run to compare to the unsaturation pattern to that of the 18:2 expected to be observed in the transformed cells.
Importantly, the cells were grown in the presence of the substrate 18:1 (Δ9) so that this could be incorporated to the membranes, as this fatty acid is not present in intact E. coli BL21.
Results
Our results indicate that, both in lipid extracts and in our membrane assays derived from cells transformed with Δ-12 desaturase, C18:2 is present, unlike in intact cells. Moreover, this 18:2 has the same unsaturation pattern as our standard, meaning the 18:2 found in the transformed cells is the expected 18:2 (Δ9,12).
Conclusion
Lipid profiles of E. coli transformed with our construct show that this Δ-12 desaturase is able to catalyze the desaturation of oleic acid to give linoleic acid. In this way, this BioBrick can be used to build a biosynthetic pathway for PUFAs and Omega-3 fatty acids, along with other desaturases and elongases.