Composite

Part:BBa_K243016

Designed by: Freiburg Bioware09   Group: iGEM09_Freiburg_bioware   (2009-10-14)
Revision as of 00:35, 22 October 2009 by Isabel (Talk | contribs)

His-FluA-Long Linker-Fok_i


This combination uses the benefits of an His tag for purification. It is also linked with a FluoresceinA tag. The Long Linker (GlySerGlyGly)x3 connects the parts and adds additional space between them to guarantee the independent function of FluA tag and the protein domain Fok_i.

Usage and Biology

This composite part is one part of our universal endonuclease and it needs another composite part like BBa_K243022to build a functional heterodimer. The FluA tag guides the part to DNA which is hybridized with a Fluorescein labeled oligonucleotide. The Long Linker creates a distance of 36bp between the FluA and the linked Fok_i protein domain. The His Tag serves as a 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 FluoresceinA tag allows the measurement by quenching and the coupling to a fluorescein linked oligo. Emanating from our 3D modeling this combination of FluA tagged oligo and the construct containing the protein domain Fok_i is more efficient than the use of a combination of DigA tagged oligo with a construct containing Fok_i. To avoid interactions between the FluA tag with the connected protein domain Fok_i we applied the Long Linker. The linker itself has no influence on the connected parts. We decided to use the Long Linker for this construct to get a longer distance between FluA tag and Fok_i to guarantee the independent function of both parts and also to prove the optimal distance between the parts. It is important that the used linker has a certain flexibility and is long enough to avoid sterical interferences between the parts. If the linker is too long it might cause the instability of the whole construct.


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
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
Parameters
n/aHis-FluA-Long Linker-Fok_i