Part:BBa_K243017

Strep-DigA-Split Linker-Fok_a

This part is the counter part to BBa_K243010, it inhabits the active cutting site of our universal endonuclease. We prefer this part because it has another combiantion of basic parts than His-FluA-Split-Fok_i, which allows us another way of purification and linkage to the labeled oligos, which were hybridized with the target DNA.[explication] The purification is made with a Streptavidin column and the linkage is done by digoxigenin binding.

Usage and Biology

This composite part is one part of our universal endonuclease and it needs another composite part like BBa_K243010 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_a protein domain. The Strep-Tag serve 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 shows also 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 that efficiently than the use of a combination of DigA tagged oligo with a construct containing Fok_a. To avoid interactions between the FluA-tag with the connected protein domain Fok_a 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 278
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1132