Difference between revisions of "Part:BBa K3128011:Design"
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<partinfo>BBa_K3128011 short</partinfo>
 
<partinfo>BBa_K3128011 short</partinfo>
  
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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.
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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.
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The plasmid was digested by the corresponding enzymes and the protein gene was inserted.
  
 
[[File:BBa K3128011 design1.png|900px|thumb|left]]
 
[[File:BBa K3128011 design1.png|900px|thumb|left]]
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===Design Notes===
 
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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===

Revision as of 17:15, 23 September 2019


OmpX-WT fused with T18 subpart of Bordetella Pertussis adenylate cyclase under lactose promoter


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.

BBa K3128011 design1.png
BBa K3128011 design2.png



Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1504
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1045
    Illegal NgoMIV site found at 1455
    Illegal AgeI site found at 1261
  • 1000
    COMPATIBLE WITH RFC[1000]


Source

pUT18 plasmid from Euromedex BACTH kit was used. OmpX gene and GGS linker were synthesized by IDT because they were unavailable on iGEM plates.

References