Difference between revisions of "Part:BBa K3114022:Design"

 
(→‎Design Notes)
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===Design Notes===
 
===Design Notes===
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When designing this circuit and the rest of our [https://2019.igem.org/Team:Calgary/Part_Collection collection], we were interested in creating parts that could be used in Golden Gate assembly right out of the distribution kit without the need to first domesticate them in a Golden Gate entry vector. As such, the basic parts are not compatible with the iGEM Type IIS RFC[1000] assembly standard because we included the BsaI restriction site and MoClo standard fusion site in the part’s sequence.
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[[Image:T--Calgary--MoClo1.png|900px|thumb|center|Figure 1. Fusion sites used in the MoClo standard for Golden Gate assembly (Weber et al., 2011).]]
  
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This part was constructed using component parts listed below. It is iGEM Type IIS RFC[1000] compatible.
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<ul>
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<li>T7 Promoter and strong RBS [https://parts.igem.org/Part:BBa_K3114012 (BBa_K3114012)]</li>
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<li>6xHis-tagged 6GIX [https://parts.igem.org/Part:BBa_K3114006 (BBa_K3114006)]</li>
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<li>Double terminator [https://parts.igem.org/Part:BBa_K3114013 (BBa_K3114013)]</li>
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</ul>
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The circuit was designed for inducible, high-level expression and has been codon optimized for <i>E. coli</i>.  A 6X Histidine affinity chromatography tag was added to the N-terminus of the 6GIX coding sequence for purification.
  
 
===Source===
 
===Source===

Revision as of 04:38, 21 October 2019


6xHis-tagged water-soluble chlorophyll binding protein (6GIX) circuit with no signal peptide


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 654
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 179
    Illegal AgeI site found at 122
    Illegal AgeI site found at 438
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1
    Illegal BsaI.rc site found at 765


Design Notes

When designing this circuit and the rest of our collection, we were interested in creating parts that could be used in Golden Gate assembly right out of the distribution kit without the need to first domesticate them in a Golden Gate entry vector. As such, the basic parts are not compatible with the iGEM Type IIS RFC[1000] assembly standard because we included the BsaI restriction site and MoClo standard fusion site in the part’s sequence.

Figure 1. Fusion sites used in the MoClo standard for Golden Gate assembly (Weber et al., 2011).

This part was constructed using component parts listed below. It is iGEM Type IIS RFC[1000] compatible.

The circuit was designed for inducible, high-level expression and has been codon optimized for E. coli. A 6X Histidine affinity chromatography tag was added to the N-terminus of the 6GIX coding sequence for purification.

Source

wip

References