Composite

Part:BBa_K4343077

Designed by: Yue Shu   Group: iGEM22_NNU-China   (2022-10-09)


pUC-HUH-IntC-FmDes12

FmDes12 gene encodes a ∆-12 desaturase from Fusarium moniliform, which can catalyze C18:1 to C18:2. This gene is expressed in Y. lipolytica.

Characterization

We constructed a plasmid containing promoter TEF, FmDes12, terminator lip2t, and homologous arm through the T4 ligase. The plasmid was linearized by enzyme cleavage and then interated into the Intc site of strain Po1f. FmDes12 gene was expressed under the control of TEF promoter. Then the activity of FmDes12 was tested by gas chromatography.

puc-huh-intc-fmdes12-map.png

Result

The enzyme activity of FmDes12 was the best, the C18:1 content decreased from 53.8% to 9.5%, and C18:2 reached 62.7% of TFA. The ratio of C18:2/C18:1 increased from 0.378 to 6.62, and the conversion of C18:1 was 81.1%. (Fig: Po1f-4)

part-5.jpg

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 6773
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 3344
    Illegal XhoI site found at 3377
    Illegal XhoI site found at 8239
    Illegal XhoI site found at 9155
    Illegal XhoI site found at 9446
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 3532
    Illegal NgoMIV site found at 6797
    Illegal AgeI site found at 1422
    Illegal AgeI site found at 1521
    Illegal AgeI site found at 1882
    Illegal AgeI site found at 4585
    Illegal AgeI site found at 4946
    Illegal AgeI site found at 6088
    Illegal AgeI site found at 7544
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1
    Illegal BsaI site found at 1425
    Illegal BsaI site found at 2062
    Illegal BsaI site found at 5126
    Illegal BsaI site found at 5663
    Illegal BsaI site found at 6091
    Illegal BsaI site found at 7547
    Illegal BsaI.rc site found at 1419
    Illegal BsaI.rc site found at 6085
    Illegal BsaI.rc site found at 7541
    Illegal BsaI.rc site found at 8041
    Illegal BsaI.rc site found at 8051
    Illegal BsaI.rc site found at 9469
    Illegal SapI site found at 8967
    Illegal SapI.rc site found at 1787
    Illegal SapI.rc site found at 4851


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