Composite

Part:BBa_K243022:Design

Designed by: Freiburg Bioware09   Group: iGEM09_Freiburg_bioware   (2009-10-18)
Revision as of 02:06, 22 October 2009 by Sarah (Talk | contribs) (→‎Design Notes)

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DsbA-Strep-DigA-Split Linker-Fok_a


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 338
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1186


Design Notes

We applied the Streptavidine tag to enable a simultaneous purification of constructs with a His tag. Strep tag also shows a higher affinity towards Strep-Tactin than His tag. For that the purification with Strep tag is more specific. The used DigoxigeninA tag allows the coupling to an fluorescein linked oligo. Emanating from our 3D modeling this combination of DigA tagged oligos and the construct containing the protein domain Fok_a is more efficient than the use of a combination of FluA tagged oligos with a construct containing Fok_a. To avoid interactions between the DigA tag with the connected protein domain Fok_a we applied the Split Linker. The Linker itself has no influence on the connected parts. We decided to use the Split Linker for this construct to get a longer distance between DigA tag and Fok_a to guarantee the independent function of both parts. This Linker is an improved part of the team Freiburg08 and it is a suitable linker for fusion proteins. The properties of the split linker are a good compromise between the stability and the distance of the connection between protein domain Fok_a and the anticalin tag. The additional linkage of the construct with DsbA allows the export of the protein into the periplasm. The accumulation of the expressed protein in the periplasm can be used for the purification of the protein 

Commented GenBank file

Source

Combined by serial cloning steps

References