Composite

Part:BBa_K243036

Designed by: Freiburg Bioware09   Group: iGEM09_Freiburg_bioware   (2009-10-20)
Revision as of 22:06, 21 October 2009 by Sarah (Talk | contribs)

His-Dig-Split Linker-Fok_a

This combination uses the benefits of a His-tag (Polyhistidin-tag) for purification. It is also linked with a DigoxigeninA tag DigA. The Split Linker connects the parts and adds additional space between them to guarantee the independent function of DigA tag and the protein domain Fok_a.


Usage and Biology

This composite part is one part of our universal endonuclease and it needs another composite part like BBa_K243010 to build a functional heterodimer. The DigA tag guides the part to the DNA which is hybridized with a Digoxigenin labeled oligonucleotide. The Split Linker creates a distance of 51bp between the DigA and the linked Fok_a protein domain. The His-Tag serves as purification tag for Ni-NTA column purification.

We applied the His tag to enable a simultaneous purification of constructs with a Strep tag. The used DigoxigeninA-tag allows the coupling to a 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.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 272
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 1126


[edit]
Categories
Parameters
n/aHis-Dig-Split Linker-Fok_a