Difference between revisions of "Part:BBa K243016"

(Usage and Biology)
 
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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.
 
  
 
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Latest revision as of 02:12, 22 October 2009

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.



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]