Part:BBa_K243015

Strep-DigA-Split Linker-Fok_i

This combination uses the benefits of a Streptavidin-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_i.

Usage and Biology

This composite part is one part of our universal endonuclease and it needs another composite part like BBa_K243024 to build a functional heterodimer. The DigA tag guides the part to DNA which is hybridized with a Digoxigenin labeled oligonucleotide. The Split Linker creates a distance of 51bp between the DigA and the linked Fok_i protein domain. The Strep Tag serves as a 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. For that the purification with Strep-tag is more specific. The used DigA tag allows the coupling to an Digoxigenin linked oligo. Emanating from our 3D modeling this combination of DigA tagged oligo and the construct containing the protein domain Fok_i is not as efficient as the use of a combination of FluA tagged oligo with a construct containing Fok_i. To avoid interactions between the DigA tag with the connected protein domain Fok_i we applied the Split Linker. This linker is an improved part of the team Freiburg08 and a suitable linker for fusion proteins.

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]