Composite

Part:BBa_K4343070

Designed by: Xiaolu Sun   Group: iGEM22_NNU-China   (2022-10-09)


TsElo5

The TsElo5 gene encodes a ∆-5 elongase from Thraustochytrium sp. that catalyzes the conversion of Eicosapentaenoic acid (EPA) to Docosapentaenoic acid (DPA), an ω-3 polyunsaturated fatty acid (PUFA).

Characterisation

We constructed a plasmid containing promoter TEF, TsELO5, and terminator Cyc1t through the T4 ligase. The plasmid was linearized by enzyme cleavage and then transformed into engineered strain Po1f-20 (22.4% of EPA in the TFAs) and randomly integrated into its genome. The TsElo5 gene was expressed in the presence of the promoter TEF, which can catalyze the conversion of Eicosapentaenoic acid (EPA; C20:5n-3) to Docosapentaenoic acid (DPA; C22:5n-3), and subsequently, the activity of TsElo5 was examined by gas chromatography.
puc-huh-tselo5-map.png

Result

In order to obtain an engineered strain for co-production of DHA and EPA production, Po1f-20 was used as the starting strain. Besides, ∆-5 elongase were identified to convert EPA to DHA, so TsElo5 gene from Thraustochytrium sp. was randomly integrated in the genome of Po1f-20 to produce Docosapentaenoic acid (DPA; C22:5n-3). The GC analysis indicated that the percentage of EPA and DPA in the engineered strain were 3.5% and 23.9% of TFAs, respectively.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 5492
    Illegal XhoI site found at 1920
    Illegal XhoI site found at 1953
    Illegal XhoI site found at 4685
    Illegal XhoI site found at 5473
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2108
    Illegal AgeI site found at 97
    Illegal AgeI site found at 458
    Illegal AgeI site found at 3161
    Illegal AgeI site found at 3522
    Illegal AgeI site found at 4664
    Illegal AgeI site found at 5625
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1
    Illegal BsaI site found at 638
    Illegal BsaI site found at 3702
    Illegal BsaI site found at 4239
    Illegal BsaI site found at 4667
    Illegal BsaI site found at 5628
    Illegal BsaI.rc site found at 4661
    Illegal BsaI.rc site found at 5622
    Illegal BsaI.rc site found at 5892
    Illegal SapI.rc site found at 363
    Illegal SapI.rc site found at 3427


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