Difference between revisions of "Part:BBa K4343072"
(→‎Characterisation)
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===Characterisation===
 
===Characterisation===
 
We constructed a plasmid containing promoter TEF, MaElo2, terminator Cyc1t, and homologous arm through the T4 ligase. The plasmid was linearized by enzyme cleavage and then transformed into Yarrowia lipolytica. This segment would be 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, MaElo2, terminator Cyc1t, and homologous arm through the T4 ligase. The plasmid was linearized by enzyme cleavage and then transformed into Yarrowia lipolytica. This segment would be 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-maelo2-map.png</center>
  
 
===Result===
 
===Result===

Revision as of 14:48, 9 October 2022


pUC-HUH-MaElo2

MaElo2 gene encodes a C16/18 elongase from Mortierella alpina, which can catalyze Palmitic acid (C16:0) to Stearic acid (C18:0). This gene is expressed in Y. lipolytica.

Characterisation

We constructed a plasmid containing promoter TEF, MaElo2, terminator Cyc1t, and homologous arm through the T4 ligase. The plasmid was linearized by enzyme cleavage and then transformed into Yarrowia lipolytica. This segment would be 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-maelo2-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. And it was found that MaElo2 with three copies (Po1f-17) showed the best result and EPA percentage of total fatty acids was 7.3%. Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 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 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 5514
  • 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 5517
    Illegal BsaI.rc site found at 4661
    Illegal BsaI.rc site found at 5511
    Illegal BsaI.rc site found at 5781
    Illegal SapI.rc site found at 363
    Illegal SapI.rc site found at 3427