Difference between revisions of "Part:BBa K4343074"
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===Characterisation===
 
===Characterisation===
 
Three copies of this gene, two copies of IgASE2 (BBa_K4343075), and two copies of EgDes8 (BBa_K4343067) were randomly integrated into the genome of the engineered strain Po1F-18. The ∆-9 elongase (EhElo9) gene was expressed under the control of promoter TEF to generate ∆-9 elongase, which catalyzes the formation of Eicosadienoic acid (EDA; C20:2n-6), the engineered strain Po1F-19 was obtained. After fermentation, the effect of the increased copy number of these three genes on the increase of EPA content was tested by gas chromatography, and 16.3% of EPA in the engineered strain Po1F-18 was obtained.
 
Three copies of this gene, two copies of IgASE2 (BBa_K4343075), and two copies of EgDes8 (BBa_K4343067) were randomly integrated into the genome of the engineered strain Po1F-18. The ∆-9 elongase (EhElo9) gene was expressed under the control of promoter TEF to generate ∆-9 elongase, which catalyzes the formation of Eicosadienoic acid (EDA; C20:2n-6), the engineered strain Po1F-19 was obtained. After fermentation, the effect of the increased copy number of these three genes on the increase of EPA content was tested by gas chromatography, and 16.3% of EPA in the engineered strain Po1F-18 was obtained.
 +
<center>https://static.igem.wiki/teams/4343/wiki/puc-huh-ehelo9-map.png</center>
  
 
===Result===
 
===Result===
−
Furthermore, EhElog9, IgASE2 and EgDes8 were randomly integrated in the genome of Po1f-18 to obtain Po1f-19. It can be found that EPA Po1f-19 produced made up 16.3% of TFAs. The results of qPCR demonstrate that the genome of Po1f-19 was integrated of 3 copies of the EnElo9, 3 copies of the IgASE2, and 2 copies of the EgDes8 (Fig. ).
+
Furthermore, EhElo9, IgASE2 and EgDes8 were randomly integrated in the genome of Po1f-18 to obtain Po1f-19. It can be found that EPA Po1f-19 produced made up 16.3% of TFAs. The results of qPCR demonstrate that the genome of Po1f-19 was integrated of 3 copies of the EnElo9, 3 copies of the IgASE2, and 2 copies of the EgDes8 (Fig: Po1f-19).
 +
<center>https://static.igem.wiki/teams/4343/wiki/many-copies.png</center>
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 12:56, 10 October 2022


pUC-HUH-EhElo9

EhElo9 encodes ∆-9 elongase from Emiliania huxleyi which functions in the Δ-9 elongase/desaturase pathway and could convert C18:2 to C20:2.

Characterisation

Three copies of this gene, two copies of IgASE2 (BBa_K4343075), and two copies of EgDes8 (BBa_K4343067) were randomly integrated into the genome of the engineered strain Po1F-18. The ∆-9 elongase (EhElo9) gene was expressed under the control of promoter TEF to generate ∆-9 elongase, which catalyzes the formation of Eicosadienoic acid (EDA; C20:2n-6), the engineered strain Po1F-19 was obtained. After fermentation, the effect of the increased copy number of these three genes on the increase of EPA content was tested by gas chromatography, and 16.3% of EPA in the engineered strain Po1F-18 was obtained.

puc-huh-ehelo9-map.png

Result

Furthermore, EhElo9, IgASE2 and EgDes8 were randomly integrated in the genome of Po1f-18 to obtain Po1f-19. It can be found that EPA Po1f-19 produced made up 16.3% of TFAs. The results of qPCR demonstrate that the genome of Po1f-19 was integrated of 3 copies of the EnElo9, 3 copies of the IgASE2, and 2 copies of the EgDes8 (Fig: Po1f-19).

many-copies.png

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 NgoMIV site found at 5341
    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 5487
  • 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 5490
    Illegal BsaI.rc site found at 4661
    Illegal BsaI.rc site found at 5484
    Illegal BsaI.rc site found at 5754
    Illegal SapI.rc site found at 363
    Illegal SapI.rc site found at 3427