Difference between revisions of "Part:BBa K4343086"
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===Characterisation===
 
===Characterisation===
 
We constructed a plasmid containing promoter TEF, MaElo2, terminator Cyc1t, and Scp2 homologous arm through the T4 ligase. The plasmid was linearized by enzyme cleavage and then was integrated into the Scp2 site of strain Po1f-3. MaElo2 gene was expressed under the control of TEF promoter, which can catalyze the elongation of C16:0 to C18:0, it also has catalytic activity for C18:1. Then the activity of MaElo2 was tested by gas chromatography (GC).
 
We constructed a plasmid containing promoter TEF, MaElo2, terminator Cyc1t, and Scp2 homologous arm through the T4 ligase. The plasmid was linearized by enzyme cleavage and then was integrated into the Scp2 site of strain Po1f-3. MaElo2 gene was expressed under the control of TEF promoter, which can catalyze the elongation of C16:0 to C18:0, it also has catalytic activity for C18:1. Then the activity of MaElo2 was tested by gas chromatography (GC).
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<center>https://static.igem.wiki/teams/4343/wiki/puc-huh-scp2-maelo2-map.png</center>
  
 
===Result===
 
===Result===

Revision as of 13:19, 9 October 2022


pUC-HUH-Scp2-MaElo2

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

Characterisation

We constructed a plasmid containing promoter TEF, MaElo2, terminator Cyc1t, and Scp2 homologous arm through the T4 ligase. The plasmid was linearized by enzyme cleavage and then was integrated into the Scp2 site of strain Po1f-3. MaElo2 gene was expressed under the control of TEF promoter, which can catalyze the elongation of C16:0 to C18:0, it also has catalytic activity for C18:1. Then the activity of MaElo2 was tested by gas chromatography (GC).

puc-huh-scp2-maelo2-map.png

Result

The C16:0 content was reduced to 10.5% of TFAs and the percentage of C18:2 they produced were almost doubled.

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 1431
    Illegal XhoI site found at 3465
    Illegal XhoI site found at 3498
    Illegal XhoI site found at 7853
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 3653
    Illegal AgeI site found at 889
    Illegal AgeI site found at 1543
    Illegal AgeI site found at 1642
    Illegal AgeI site found at 2003
    Illegal AgeI site found at 4706
    Illegal AgeI site found at 5067
    Illegal AgeI site found at 6209
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1
    Illegal BsaI site found at 1546
    Illegal BsaI site found at 2183
    Illegal BsaI site found at 5247
    Illegal BsaI site found at 5784
    Illegal BsaI site found at 6212
    Illegal BsaI site found at 7062
    Illegal BsaI.rc site found at 401
    Illegal BsaI.rc site found at 1540
    Illegal BsaI.rc site found at 6206
    Illegal BsaI.rc site found at 7056
    Illegal BsaI.rc site found at 7326
    Illegal BsaI.rc site found at 8488
    Illegal BsaI.rc site found at 8872
    Illegal SapI.rc site found at 1908
    Illegal SapI.rc site found at 4972