Part:BBa_K243018

His-DigA-Split Linker-Fok_i

This combination uses the benefits of an His-tag (Polyhistidin-tag) for purification. It is also linked with a DigoxigenineA-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 built to a functional heterodimer. The DigA tag leads the part to DNA which is hybridized with an Digoxigenin labeled oligonucleotide. The split linker makes a distance from 51bp between the DigA and the linked Fok_i protein domain. The His-Tag serve as 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 the coupling to an flurescein linked oligo. Emanating from our 3D modeling this combination of DigA tagged oligos and the construct containing the protein domain Fok_i is not that efficiently than the use of a combination of FluA tagged oligos 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. The Linker itself has no influence of the connected parts. We decided to use the Split Linker for this construct to get a longer distance between DigA-tag and Fok_i to guarantee the independent function of both parts. This Linker is an improved part of the team Freiburg08 and it is 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 272
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]