Team:CityU HK/project/result desaturase


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Cloning and Expression of Δ9-Δ12-Δ15 Desaturase Genes

Overview :

In an attempt to overexpress the Δ9-Δ12-Δ15 desaturases gene cassette in E.coli to convert stearic acid into ALA, we subcloned the Δ9-Δ12-Δ15 desaturase gene cluster downstream of the LacI promoter in the vector pSB1C3 (of BioBrick BBa_R0010). We succeeded in subcloning the desaturase gene cassette into the plasmid pSB1A2 (which is supposedly “promoterless”) but not pSB1C3. Nonetheless, expression of the Δ9-Δ12-Δ15 desaturase gene cluster in recombinant E. coli was measured by quantitative RT-PCR (qRT-PCR) and we also attempted to determine the effect of Δ9-Δ12-Δ15 desaturase gene expression on intracellular concentrations of oleic acid and α-linolenic acid (ALA) in control and recombinant E. coli cells by GC-MS.

Results :

Quantitative real-time PCR analysis showed that the Δ9-12Δ-Δ15 desaturase mRNA transcripts were expressed at high levels in the recombinant E. coli cells as compared to the control (Figure 1).

Figure 1. Quantitative RT-PCR analysis of desaturase genes. Expression of Δ9-Δ12-Δ15 desaturase genes in control and recombinant E. coli TOP10 cells. E. coli cells were cultured in LB + stearic acid (0.2 mM) and harvested at 0 h, 4 h and 6h for qRT-PCR and GC-MS analyses.

For GC-MS analysis, no oleic acid or ALA was detected in both the control and recombinant E. coli cells (data not shown). However, due to the lack of an ALA standard, the results need to be further confirmed in future experiments to determine if stearic acid is converted to ALA in the Δ9-Δ12-Δ15 desaturase recombinant E. coli. Moreover, due to time constraint, these experiments were performed only once, and further replicates will carried out in future studies in order to determine the statistical significance of the data.

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