Difference between revisions of "Part:BBa K3128024:Design"

(→‎Design Notes)
 
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===Design Notes===
 
===Design Notes===
 +
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>
 +
 +
<br>
 
Euromedex BACTH kit contains the pUT18 plasmid with a MCS cassette followed by the T18 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.
 
Euromedex BACTH kit contains the pUT18 plasmid with a MCS cassette followed by the T18 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===
 
===Source===
  
−
pUT18-ZIP plasmid from Euromedex BACTH kit was used (containing the T18 subpart and the leucine zipper gene). OmpX gene and GGS linker were synthesized by IDT because they were unavailable on iGEM plates.
+
T18 is from <i>bordetella pertussis</i><br>
 +
Ompx is from <i> Escherichia coli</i> <br>
 +
<i> pUT18 </i> plasmid from Euromedex BACTH kit was used (containing the T18 subpart and the leucine zipper gene).<br>
 +
OmpX gene and GGS linker were synthesized by IDT because they were unavailable on iGEM plates.
 +
 
  
 
===References===
 
===References===
 +
[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 20:45, 9 October 2019


COMP fused with T18 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 1601
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1142
    Illegal NgoMIV site found at 1552
    Illegal AgeI site found at 1358
  • 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 pUT18 plasmid with a MCS cassette followed by the T18 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
pUT18 plasmid from Euromedex BACTH kit was used (containing the T18 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