Difference between revisions of "Part:BBa K4343073"
(→‎Characterisation)
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===Characterisation===
 
===Characterisation===
 
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 transformed into Yarrowia lipolytica. The segment was randomly integrated into its genome. FmDes12 gene was expressed under the control of TEF promoter. Then the activity of FmDes12 was tested by gas chromatography.
 
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 transformed into Yarrowia lipolytica. The segment was randomly integrated into its genome. FmDes12 gene was expressed under the control of TEF promoter. Then the activity of FmDes12 was tested by gas chromatography.
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<center>https://static.igem.wiki/teams/4343/wiki/puc-huh-fmdes12-map.png</center>
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===Result===
 
===Result===
 
In order to reduce the amount of intermediates and increase the amount of EPA, elongases/desaturases above were randomly integrated of several copies in the genome. Besides, the optimal copy numbers of the elongases/desaturases were detected by qPCR. Firstly, MaElo2 was randomly integrated into the Po1f-3 genome because the content of C16:0 accounted for about 15% of the TFAs of strain Po1f-3. It was found that MaElo2 with three copies (Po1f-17) showed the best result and EPA percentage of total fatty acids it produced was 7.3%. Similarly, Po1f-18 was obtained by integrating two copies of FmDes12 and the proportion of EPA was 9.6% (Fig. ).
 
In order to reduce the amount of intermediates and increase the amount of EPA, elongases/desaturases above were randomly integrated of several copies in the genome. Besides, the optimal copy numbers of the elongases/desaturases were detected by qPCR. Firstly, MaElo2 was randomly integrated into the Po1f-3 genome because the content of C16:0 accounted for about 15% of the TFAs of strain Po1f-3. It was found that MaElo2 with three copies (Po1f-17) showed the best result and EPA percentage of total fatty acids it produced was 7.3%. Similarly, Po1f-18 was obtained by integrating two copies of FmDes12 and the proportion of EPA was 9.6% (Fig. ).

Revision as of 14:42, 9 October 2022


pUC-HUH-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.

Characterisation

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 transformed into Yarrowia lipolytica. The segment was randomly integrated into its genome. FmDes12 gene was expressed under the control of TEF promoter. Then the activity of FmDes12 was tested by gas chromatography.

puc-huh-fmdes12-map.png

Result

In order to reduce the amount of intermediates and increase the amount of EPA, elongases/desaturases above were randomly integrated of several copies in the genome. Besides, the optimal copy numbers of the elongases/desaturases were detected by qPCR. Firstly, MaElo2 was randomly integrated into the Po1f-3 genome because the content of C16:0 accounted for about 15% of the TFAs of strain Po1f-3. It was found that MaElo2 with three copies (Po1f-17) showed the best result and EPA percentage of total fatty acids it produced was 7.3%. Similarly, Po1f-18 was obtained by integrating two copies of FmDes12 and the proportion of EPA was 9.6% (Fig. ).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 5349
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1920
    Illegal XhoI site found at 1953
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2108
    Illegal NgoMIV site found at 5373
    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 6120
  • 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 6123
    Illegal BsaI.rc site found at 4661
    Illegal BsaI.rc site found at 6117
    Illegal BsaI.rc site found at 6617
    Illegal BsaI.rc site found at 6627
    Illegal SapI.rc site found at 363
    Illegal SapI.rc site found at 3427