Difference between revisions of "Part:BBa K925000"
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Importantly, the cells were grown in the presence of manually added substrate 18:1 (Δ9). 18:1 fatty acids are not present in unmodified <i>E. coli</i> BL21. | Importantly, the cells were grown in the presence of manually added substrate 18:1 (Δ9). 18:1 fatty acids are not present in unmodified <i>E. coli</i> BL21. | ||
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[[Image:FAME-_GC_MS_STANDARDSc.png]] | [[Image:FAME-_GC_MS_STANDARDSc.png]] | ||
Revision as of 22:37, 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 Δ12 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). This converts the substrate into linoleic acid (18:2 Δ9,12), a polyunsaturated fatty acid (PUFA). Mass spectrometry results show that feeding oleic acid to transformed E. coli, the bacterial membrane composition changes. 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. Our samples were membrane assays and lipid extracts from E. coli expressing this desaturase.
As a control, the same FAME-GC analysis was performed on unmodified cells, and the lipid profiles were compared. FAME 18:2 (Δ9,12) standard was also run to compare the desaturation pattern to the expected 18:2 of the transformed cells.
Importantly, the cells were grown in the presence of manually added substrate 18:1 (Δ9). 18:1 fatty acids are not present in unmodified E. coli BL21.
Results
Our results indicate that C18:2 is present in both the lipid extracts and membrane assays from cells transformed with the Δ-12 desaturase, unlike in untransformed cells. Moreover, this 18:2 has the same desaturation pattern as the control, 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.