Difference between revisions of "Part:BBa K4343067"

 
 
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The EgDes8 gene encodes a ∆-8 desaturase from Euglena gracilis that catalyzes the conversion of eicosadienoic acid (EDA; C20: 2n-6) to dihomo-γ-linolenic acid (DGLA; C20: 3n-6).
 
The EgDes8 gene encodes a ∆-8 desaturase from Euglena gracilis that catalyzes the conversion of eicosadienoic acid (EDA; C20: 2n-6) to dihomo-γ-linolenic acid (DGLA; C20: 3n-6).
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===Characterisation===
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We constructed a plasmid containing promoter TEF, EgDes8, and terminator Cyc1t through the T4 ligase. The plasmid was linearized by enzyme cleavage and then transformed into engineered Yarrowia lipolytica and randomly integrated into its genome. The engineered strain Po1f-1 was obtained, which can catalyze the conversion of eicosadienoic acid (EDA; C20: 2n-6) to dihomo-γ-linolenic acid (DGLA; C20: 3n-6) by expressing EgDes8 gene. And subsequently, the activity of EgDes8 was examined by gas chromatography.<br>
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https://static.igem.wiki/teams/4343/wiki/puc-huh-egdes8-map.png
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===Result===
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We integrate one copy of codon-optimized EgElo8 gene from Euglena gracilis under the control of strong promoter PTEF into the genome to obtain the engineered strain Po1f-1 by non-homologous End Joining (NHEJ). The percentage of eicosadienoic acid (EDA; C20: 2n-6) and DGLA were 20.8% and 8.5%, respectively(fig:po1f-1).<br>
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https://static.igem.wiki/teams/4343/wiki/part-3.jpg
  
 
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Latest revision as of 10:01, 9 October 2022


pUC-HUH-EgDes8

The EgDes8 gene encodes a ∆-8 desaturase from Euglena gracilis that catalyzes the conversion of eicosadienoic acid (EDA; C20: 2n-6) to dihomo-γ-linolenic acid (DGLA; C20: 3n-6).

Characterisation

We constructed a plasmid containing promoter TEF, EgDes8, and terminator Cyc1t through the T4 ligase. The plasmid was linearized by enzyme cleavage and then transformed into engineered Yarrowia lipolytica and randomly integrated into its genome. The engineered strain Po1f-1 was obtained, which can catalyze the conversion of eicosadienoic acid (EDA; C20: 2n-6) to dihomo-γ-linolenic acid (DGLA; C20: 3n-6) by expressing EgDes8 gene. And subsequently, the activity of EgDes8 was examined by gas chromatography.
puc-huh-egdes8-map.png

Result

We integrate one copy of codon-optimized EgElo8 gene from Euglena gracilis under the control of strong promoter PTEF into the genome to obtain the engineered strain Po1f-1 by non-homologous End Joining (NHEJ). The percentage of eicosadienoic acid (EDA; C20: 2n-6) and DGLA were 20.8% and 8.5%, respectively(fig:po1f-1).
part-3.jpg

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 4994
    Illegal XhoI site found at 1920
    Illegal XhoI site found at 1953
    Illegal XhoI site found at 5315
  • 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 5955
  • 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 5958
    Illegal BsaI.rc site found at 4661
    Illegal BsaI.rc site found at 5952
    Illegal BsaI.rc site found at 6222
    Illegal SapI.rc site found at 363
    Illegal SapI.rc site found at 3427
    Illegal SapI.rc site found at 4939