Part:BBa_K243025

Strep-FluA-Middle Linker-Fok_i

This combination uses the benefits of a Streptavidin tag for purification. It is also linked with a FluoresceinA-tag. The Middle Linker (GlySerGlyGly)x2 connects the parts and adds additional space between them to guarantee the independent function of FluA 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_K243017 to build a functional heterodimer. The FluA tag guides the part to DNA which is hybridized with a Fluorescein labeled oligonucleotide. The middle linker creates a distance of 24bp between the FluA and the linked Fok_i protein domain. The StrepTag serves as purification tag for Streptavidin column purification.

We applied the Streptavidine tag to enable a simultaneous purification of constructs with a His tag. Strep tag also shows a higher affinity towards Strep-Tactin than His tag. In this case the purification with Strep tag is more specific. 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, whereas the combination of DigA tagged oligo and constructs consisting Fok_a seems to be more efficient. To avoid interactions between the FluA tag with the connected protein domain Fok_i we applied the Middle Linker. The Linker itself has no influence on the connected parts. We decided to use the Middle Linker for this construct 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 steric interferences between the parts. If the linker is too long it might cause a 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 278
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]