Part:BBa_K243014

His-FluA-Middle Linker-Fok_i

This combination uses the benefits of an His-tag for purification. It is also linked with a FluroescineA-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_i.

Usage and Biology

This composite part is one part of our universal endonuclease and it needs another composite part like BBa_K243017 to built to a functional heterodimer. The FluA tag leads the part to DNA which is hybridized with an Fluorescein labeled oligonucleotide. The middle linker makes a distance from 24bp between the FluA and the linked Fok_i protein domain. The His-tag serves as purification tag for a Ni-NTA column purification.

We applied the His-tag to enable a simultaneous purification of constructs with a Strep-tag. The used FluroescineA-tag allows the measurement by quenching and the coupling to an flurescein 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. To avoid interactions between the FluA-tag with the connected protein domain Fok_i we applied the Middel Linker. The Linker itself has no influence of 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 272
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]