Difference between revisions of "Part:BBa K925001"
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===Characterisation===
 
===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 well as to Δ -12 desaturase. This was so that the product of Δ-12 desaturase (18:2 ; Δ9,12) could be used by Δ-15 for the formation of  18:3 ; Δ9,12,15.
 +
 +
Importantly, the cells were grown in the presence of Δ12 desaturase’s 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.
 +
 +
As a control the same FAME-GC analysis was performed in intact cells, and the lipid profiles were compared. Additionally, FAME 18:3 (Δ9,12,15) standard was run to compare to the unsaturation pattern to that of the 18:3 expected to be observed in the transformed cells.
 +
  
 
===Results===
 
===Results===
 +
Our results indicate that, both in lipid extracts and in our membrane assays derived from cells transformed with Δ-12 and Δ-15 desaturase, C18:3 is present, unlike in intact cells. Moreover, this 18:3 has the same unsaturation pattern as our standard, meaning the 18:3 found in the transformed cells is the expected 18:3 (Δ9,12,15).
  
 
===Conclusion===
 
===Conclusion===
 +
Lipid profiles of E. coli transformed with our construct show that this Δ-15 desaturase is able to catalyze the desaturation of linoleic acid to give alpha-linoleic acid, an ω-3 fatty acid. In this way, this BioBrick can be used with BBa_K925000 to build a biosynthetic pathway for PUFAs and ω -3 fatty acids, along with other desaturases and elongases.

Revision as of 21:38, 24 September 2012

Delta 15 desaturase

Short description

Delta-15 desaturase involved in an ω-3 biosynthetic pathway.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Description

This part encodes a delta-15 desaturase derived from Synechocystis sp PCC 6803. The enzyme is able to introduce a double bond at the Δ-15 site in the hydrocarbon chain of linoleic acid (18:2 ; Δ9,12), to give alpha- linoleic acid (18:3 ; Δ9,12,15), an ω -3 polyunsaturated fatty acid (PUFA). Mass spectrometry results show that when feeding oleic acid to E. coli transformed with this part in addition to Δ -12 desaturase (BBa_K925000), alpha-linoleic acid is observed in membrane lipids.

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 well as to Δ -12 desaturase. This was so that the product of Δ-12 desaturase (18:2 ; Δ9,12) could be used by Δ-15 for the formation of 18:3 ; Δ9,12,15.

Importantly, the cells were grown in the presence of Δ12 desaturase’s 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.

As a control the same FAME-GC analysis was performed in intact cells, and the lipid profiles were compared. Additionally, FAME 18:3 (Δ9,12,15) standard was run to compare to the unsaturation pattern to that of the 18:3 expected to be observed in the transformed cells.


Results

Our results indicate that, both in lipid extracts and in our membrane assays derived from cells transformed with Δ-12 and Δ-15 desaturase, C18:3 is present, unlike in intact cells. Moreover, this 18:3 has the same unsaturation pattern as our standard, meaning the 18:3 found in the transformed cells is the expected 18:3 (Δ9,12,15).

Conclusion

Lipid profiles of E. coli transformed with our construct show that this Δ-15 desaturase is able to catalyze the desaturation of linoleic acid to give alpha-linoleic acid, an ω-3 fatty acid. In this way, this BioBrick can be used with BBa_K925000 to build a biosynthetic pathway for PUFAs and ω -3 fatty acids, along with other desaturases and elongases.