Composite

Part:BBa_K243036

Designed by: Freiburg Bioware09   Group: iGEM09_Freiburg_bioware   (2009-10-20)
Revision as of 01:18, 22 October 2009 by Timo (Talk | contribs) (→‎Application)

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 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 DigoxigeninA tag allows 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 suitable 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.


Application

At least we succeeded to produce a working universal endonuclease with this part in combination with the part BBa_K243010, by cotransformation into E.coli(XL1blue). The part BBa_K243010 was cloned into our expression vector BBa_K243033 and this part was cloned into the vector pJS419 BBa_K243035. The made a cutting assay with cleared cell lysate and DNA hybridized with oligonucleotides labeled with fluorescence molecule(Cy3).


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