Difference between revisions of "Part:BBa K3629016:Design"

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===Design Notes===
 
===Design Notes===
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There is an EcoRI site within the XRP2 terminator making this part RFC10 incompatible. However, we added the BioBrick prefix and suffix so that the other enzymes (NotI, XbaI, SpeI, and PstI) could be used to clone this part into an iGEM plasmid or another plasmid. This part can also be cloned through RFC1000 assembly.
  
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The promoter [https://parts.igem.org/Part:BBa_K3629001 (BBa_K3629001)] is an intronic promoter, however the wild-type sequence contains BsaI, PstI, and SpeI restriction sites in the promoter section before the intron sequence. Therefore, single-nucleotides changes that match the ones in the functional [https://parts.igem.org/Part:BBa_K2983050 BBa_K2983050] promoter were made to remove theses sites.
  
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The native signal peptide from <I>T. reesei</I> was removed so it would not interfere with the fused Lip2 secretion tag native to <i>Y. lipolytica.</i>
  
 
===Source===
 
===Source===
  
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The coding sequence of the EGI is from <i>Trichoderma reesei</i>, however it has been codon-optimized and mutated to have optimal activity in <i>Yarrowia lipolytica<i> based on our modelling. The promoter and terminator sequences are from wild-type <i>Y. lipolytica.</i>
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The coding sequence of the EGI is from <i>Trichoderma reesei</i>, however it has been codon-optimized and mutated to have optimal activity in <i>Yarrowia lipolytica</i> based on our modelling. The promoter and terminator sequences are from wild-type <i>Y. lipolytica.</i>
  
 
===References===
 
===References===

Latest revision as of 04:45, 19 October 2020


Modified T. reesei EGI expression construct with gibson homology sequences and 6X His tag


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 1
    Illegal suffix found in sequence at 2385
    Illegal EcoRI site found at 2204
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1
    Illegal EcoRI site found at 2204
    Illegal NheI site found at 81
    Illegal SpeI site found at 2386
    Illegal PstI site found at 2400
    Illegal NotI site found at 7
    Illegal NotI site found at 2393
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1
    Illegal EcoRI site found at 2204
    Illegal BamHI site found at 2334
    Illegal XhoI site found at 132
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 1
    Illegal suffix found in sequence at 2386
    Illegal EcoRI site found at 2204
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 1
    Illegal EcoRI site found at 2204
    Illegal XbaI site found at 16
    Illegal SpeI site found at 2386
    Illegal PstI site found at 2400
    Illegal NgoMIV site found at 1326
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

There is an EcoRI site within the XRP2 terminator making this part RFC10 incompatible. However, we added the BioBrick prefix and suffix so that the other enzymes (NotI, XbaI, SpeI, and PstI) could be used to clone this part into an iGEM plasmid or another plasmid. This part can also be cloned through RFC1000 assembly.

The promoter (BBa_K3629001) is an intronic promoter, however the wild-type sequence contains BsaI, PstI, and SpeI restriction sites in the promoter section before the intron sequence. Therefore, single-nucleotides changes that match the ones in the functional BBa_K2983050 promoter were made to remove theses sites.

The native signal peptide from T. reesei was removed so it would not interfere with the fused Lip2 secretion tag native to Y. lipolytica.

Source

The coding sequence of the EGI is from Trichoderma reesei, however it has been codon-optimized and mutated to have optimal activity in Yarrowia lipolytica based on our modelling. The promoter and terminator sequences are from wild-type Y. lipolytica.

References