Difference between revisions of "Part:BBa K4343069"
 
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===Characterisation===
 
===Characterisation===
  
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We constructed a plasmid containing promoter TEF, PaDes17, 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-2 was obtained. The PaDes17 gene was expressed in the presence of the promoter TEF, which can catalyze the conversion of arachidonic acid (ARA; C20:4 n-6) to Eicosapentaenoic acid (EPA). The activity of PaDes17 was examined by gas chromatography.
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We constructed a plasmid containing promoter TEF, PaDes17, 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-2 was obtained. The PaDes17 gene was expressed in the presence of the promoter TEF, which can catalyze the conversion of arachidonic acid (ARA; C20:4 n-6) to Eicosapentaenoic acid (EPA). The activity of PaDes17 was examined by gas chromatography.<br>
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https://static.igem.wiki/teams/4343/wiki/puc-huh-pades17-leu-map.png
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<center>https://static.igem.wiki/teams/4343/wiki/puc-huh-pades17-leu-map.png</center>
  
 
===Result===
 
===Result===
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We integrate one copy of codon-optimized PaDes17 gene from Euglena gracilis under the control of strong promoter PTEF into the genome of engineered strain with one copy of EgDes5 to obtain the engineered strain Po1f-1 by non-homologous end joining (NHEJ) Fig. 1 and Fig. 2 showed that the engineered strain Po1f-2 was able to produce 16.3% of arachidonic acid (ARA; C20:4 n-6) and 2.4% of EPA in the TFAs, which enabled the synthesis of EPA in the Po1f.
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We integrate one copy of codon-optimized PaDes17 gene from Euglena gracilis under the control of strong promoter PTEF into the genome of engineered strain with one copy of EgDes5 to obtain the engineered strain Po1f-1 by non-homologous end joining (NHEJ) Fig(po1f-2) showed that the engineered strain Po1f-2 was able to produce 16.3% of arachidonic acid (ARA; C20:4 n-6) and 2.4% of EPA in the TFAs, which enabled the synthesis of EPA in the Po1f.<br>
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<center>https://static.igem.wiki/teams/4343/wiki/part-3.jpg</center>
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Latest revision as of 14:02, 9 October 2022


pUC-HUH-PaDes17(LEU)

The PaDes17 gene encodes a ∆-17 elongase from Pythium aphanidermatum that catalyzes the conversion of arachidonic acid (ARA; C20:4 n-6) to Eicosapentaenoic acid (EPA), an ω 3 polyunsaturated fatty acid (PUFA).

Characterisation

We constructed a plasmid containing promoter TEF, PaDes17, 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-2 was obtained. The PaDes17 gene was expressed in the presence of the promoter TEF, which can catalyze the conversion of arachidonic acid (ARA; C20:4 n-6) to Eicosapentaenoic acid (EPA). The activity of PaDes17 was examined by gas chromatography.

puc-huh-pades17-leu-map.png

Result

We integrate one copy of codon-optimized PaDes17 gene from Euglena gracilis under the control of strong promoter PTEF into the genome of engineered strain with one copy of EgDes5 to obtain the engineered strain Po1f-1 by non-homologous end joining (NHEJ) Fig(po1f-2) showed that the engineered strain Po1f-2 was able to produce 16.3% of arachidonic acid (ARA; C20:4 n-6) and 2.4% of EPA in the TFAs, which enabled the synthesis of EPA in the Po1f.

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 2102
    Illegal XhoI site found at 667
    Illegal XhoI site found at 696
    Illegal XhoI site found at 1844
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1239
    Illegal AgeI site found at 1667
    Illegal AgeI site found at 2769
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1
    Illegal BsaI site found at 1242
    Illegal BsaI site found at 1670
    Illegal BsaI site found at 2772
    Illegal BsaI.rc site found at 1236
    Illegal BsaI.rc site found at 1664
    Illegal BsaI.rc site found at 2766
    Illegal BsaI.rc site found at 3036
    Illegal SapI site found at 1078