Difference between revisions of "Part:BBa K3128028:Design"
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===Design Notes===
 
===Design Notes===
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https://static.igem.org/mediawiki/parts/thumb/3/3d/T--Grenoble-Alpes--bba28.1.jpg/800px-T--Grenoble-Alpes--bba28.1.jpg.png <br>
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https://static.igem.org/mediawiki/parts/thumb/f/fa/T--Grenoble-Alpes--bba28.2.jpg/800px-T--Grenoble-Alpes--bba28.2.jpg.png <br>
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EcoRI and PstI were removed after cloning by side directed mutagenesis in order to keep the RFC[10] compatibility.<br>
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https://static.igem.org/mediawiki/parts/thumb/c/c8/T--Grenoble-Alpes--bba28.3.jpg/800px-T--Grenoble-Alpes--bba28.3.jpg.png <br>
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This biobrick has a '''TAG codon''' (Y121F' mutation) inside '''OmpX''' (amber TAG) sequene which can be used to introduce any '''Non Natural Amino acid''' outside the cell ([https://2019.igem.org/Team:Grenoble-Alpes more informations]).<br>
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<br>
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The signal peptide has a conformation enabling the protein addressing to the membrane.<br>
 
The signal peptide has a conformation enabling the protein addressing to the membrane.<br>
 
This sequence is cut after the translocation.<br>  
 
This sequence is cut after the translocation.<br>  
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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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If the COMP gene is used with the signal peptide, the leucine zipper gene is present before the signal peptide, thus generating a cut in the fusion protein LeucineZipper-COMP.<br>
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<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.
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===Source===
 
===Source===
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<i> pUT18 </i> plasmid from Euromedex BACTH kit was used (containing the T18 subpart and the leucine zipper gene).<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.
 
OmpX gene and GGS linker were synthesized by IDT because they were unavailable on iGEM plates.
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 +
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===Reference===
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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 21:38, 17 October 2019


COMP fused with Leucine Zipper and T18 subpart of B.Pertussis AC under constitutive promoter


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1436
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 977
    Illegal NgoMIV site found at 1387
    Illegal AgeI site found at 1193
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 179


Design Notes

800px-T--Grenoble-Alpes--bba28.1.jpg.png
800px-T--Grenoble-Alpes--bba28.2.jpg.png
EcoRI and PstI were removed after cloning by side directed mutagenesis in order to keep the RFC[10] compatibility.
800px-T--Grenoble-Alpes--bba28.3.jpg.png

This biobrick has a TAG codon (Y121F' mutation) inside OmpX (amber TAG) sequene which can be used to introduce any Non Natural Amino acid outside the cell (more informations).

The signal peptide has a conformation enabling the protein addressing to the membrane.
This sequence is cut after the translocation.
If the COMP gene is used with the signal peptide, the leucine zipper gene is present before the signal peptide, thus generating a cut in the fusion protein LeucineZipper-COMP.


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.


Reference

EUROMEDEX BACTH