Tag

Part:BBa_K2586006

Designed by: Janek Meißner   Group: iGEM18_Goettingen   (2018-09-13)


Strep-Tag-aroA: aroA linked to a Strep-Tag

This part is the combination of (BBa_K2586007) with a Strep-Tag.
The Strep-Protein is useful as it allows the purification of the tagged protein, to carry out further interaction studies. The Strep-protein can also be used in (SPINE): Strep-protein interaction experiments. SPINE is a powerful tool to detect protein-protein interactions in vivo (Herzberg et. al. 2007). The bait protein (aroA in this case) has to be expressed with a Strep-tag under the conditions when the potential interaction partners are presumably present in the cells. The addition of formaldehyde (FA), which easily permeates the cell wall and membrane, links the bait protein to its potential interaction partners.
The bait protein together with its potential interaction partners can then be isolated using a Strep-Tactin sepharose column by eluting with desthiobiotin- or biotin-containing buffer.
The cross-links between the bait protein and the potential interaction partner can be cleaved by heating the samples in Laemmli buffer. Finally, the co-purified interaction partner(s) can be separated by [http://www.ruf.rice.edu/~bioslabs/studies/sds-page/gellab2.html (SDS PAGE)], identified in mass spectrometry and Western Blotting.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 1
    Illegal suffix found in sequence at 1387
    Illegal EcoRI site found at 74
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1
    Illegal EcoRI site found at 74
    Illegal NheI site found at 24
    Illegal SpeI site found at 1388
    Illegal PstI site found at 1402
    Illegal NotI site found at 7
    Illegal NotI site found at 1395
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1
    Illegal EcoRI site found at 74
    Illegal BamHI site found at 95
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 1
    Illegal suffix found in sequence at 1388
    Illegal EcoRI site found at 74
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 1
    Illegal EcoRI site found at 74
    Illegal XbaI site found at 16
    Illegal SpeI site found at 1388
    Illegal PstI site found at 1402
    Illegal AgeI site found at 448
    Illegal AgeI site found at 889
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 60
    Illegal BsaI.rc site found at 1123


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