Difference between revisions of "Part:BBa K925000"

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===Description===
 
===Description===
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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 <i>E. coli</i>, the bacterial membrane composition changes. Linoleic acid is observed.
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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.
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===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. Our samples were membrane assays and lipid extracts from <i>E. coli</i> expressing this desaturase.
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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.
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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.
  
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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.  
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[[Image:D16.png]]
  
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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.
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[[Image:FAME-_GC_MS_STANDARDS.png]]
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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.
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[[Image:D16a.png]]
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[[Image:FAME-_GC_MS_STANDARDSa.png]]
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===Results===
 
===Results===
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Our results indicate that C18:2 is present in both the lipid extracts and membrane assays from cells transformed with the Δ-12 desaturase. 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).  
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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).  
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===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 21:54, 24 September 2012

Delta-12 desaturase

Short description

Delta-12 desaturase involved in an Omega-3 biosynthetic pathway.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 426
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE 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.

D16.png

FAME- GC MS STANDARDS.png

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. 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.