Difference between revisions of "Part:BBa K3128023:Design"

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<partinfo>BBa_K3128023 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K3128023 SequenceAndFeatures</partinfo>
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===Design Notes===
 
===Design Notes===
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The signal peptide has a conformation enabling the protein addressing to the membrane. This sequence is cut after the translocation. If the OmpX gene is used without changes, the leucine zipper gene is present before the signal peptide, thus generating a cut in the fusion protein leucine-zipper-OmpX.  
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The signal peptide has a conformation enabling the protein addressing to the membrane.<br>
 +
This sequence is cut after the translocation. <br>
 +
If the OmpX gene is used without changes, the leucine zipper gene is present before the signal peptide, thus generating a cut in the fusion protein leucine-zipper-OmpX.<br>
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<br>
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Euromedex BACTH kit contains the pKT25 plasmid with a MCS cassette followed by the T25 subpart of the Bordetella pertussis adenylate cyclase. It allows the cloning of the protein of interest in fusion with the T18 subpart. To respect RFC rules, the plasmid has been amplificated by PCR using primers that bind from either site of the MCS cassette to eliminate it. Those primers contain also restriction sites enabling the cloning of the protein in the system. The plasmid was digested by the corresponding enzymes and the protein gene was inserted.
  
  
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===Source===
 
===Source===
  
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pKT25-ZIP plasmid from Euromedex BACTH kit was used (containing the T25 subpart and the leucine zipper gene). OmpX gene and GGS linker were synthesized by IDT because they were unavailable on iGEM plates.
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T18 is from <i>bordetella pertussis</i><br>
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Ompx is from <i> Escherichia coli</i> <br>
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<i> pKT25 </i> plasmid from Euromedex BACTH kit was used (containing the T25 subpart and the leucine zipper gene).<br>
 +
OmpX gene and GGS linker were synthesized by IDT because they were unavailable on iGEM plates.
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===References===
 
===References===
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[https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=3&ved=2ahUKEwiw4qGr_ozlAhULA2MBHZNsBdUQFjACegQIBRAC&url=http%3A%2F%2Fstatic.bioport.cn%2Fdata%2Fupload%2Fproduct%2Fspecification%2F396%2F1342594983673_396560.pdf&usg=AOvVaw2TFscbzRiSzNjAvyMsZDHY EUROMEDEX BACTH]

Latest revision as of 16:16, 8 October 2019


OmpX-WT with Leucine Zipper and T25 subpart of Bordetella Pertussis AC under lactose promoter


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1764
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 344



Design Notes

The signal peptide has a conformation enabling the protein addressing to the membrane.
This sequence is cut after the translocation.
If the OmpX gene is used without changes, the leucine zipper gene is present before the signal peptide, thus generating a cut in the fusion protein leucine-zipper-OmpX.


Euromedex BACTH kit contains the pKT25 plasmid with a MCS cassette followed by the T25 subpart of the Bordetella pertussis adenylate cyclase. It allows the cloning of the protein of interest in fusion with the T18 subpart. To respect RFC rules, the plasmid has been amplificated by PCR using primers that bind from either site of the MCS cassette to eliminate it. Those primers contain also restriction sites enabling the cloning of the protein in the system. The plasmid was digested by the corresponding enzymes and the protein gene was inserted.


Source

T18 is from bordetella pertussis
Ompx is from Escherichia coli
pKT25 plasmid from Euromedex BACTH kit was used (containing the T25 subpart and the leucine zipper gene).
OmpX gene and GGS linker were synthesized by IDT because they were unavailable on iGEM plates.


References

EUROMEDEX BACTH