Part:BBa_K243021

DsbA-His-DigA-Split Linker-Fok_a

This composite part is based on the active Fok fusion construct which is one half of the universal endonuclease, please visit this parts page for further information. In addition, the DsbA signal sequence is present to transfer the produced fusion protein into the periplasm. A Polyhistidin-tag enables easy purification of the protein after the content of the periplasm has been extracted from the induced cells.

Usage and Biology

This composite part is one part of our universal endonuclease and needs another composite part like BBa_K243010 to build a heterodimer functional as a restriction enzyme. The DigA guides the protein to DNA which is hybridized with a Digoxigenin labeled oligonucleotide. The Split Linker creates a distance of 17 aa between the DigA and the linked Fok_a protein domain. The His Tag serves as a purification tag for Ni-NTA columns.

We applied the His tag to enable purification of the expressed construct using a Ni-NTA column. The used DigoxigeninA-tag allows targeting of the protein to fluorescein-linked synthetic oligonucleotides. According to our 3D modeling, this combination of DigA-tagged oligos and the construct containing the protein domain Fok_a is more efficient than the use of a combination of FluA tagged oligos with a construct containing Fok_a. To avoid disturbing interactions between the DigA tag with the connected protein domain Fok_a, we applied the Split Linker. The amino acid composition of the Linker should assure that it has no influence on the connected parts. Compared to the short and middle linkers (visit our parts page for details), we decided to use the Split Linker for this construct to create a longer distance between DigA tag and Fok_a to guarantee the independent function of both parts. This Linker is an improved part of the team Freiburg08 and should be suitable for many fusion proteins. The properties of the split linker are a good compromise between stability and distance of the connection between protein domain Fok_a and the anticalin. The additional linkage of the construct with the DsbA signal allows the export of the protein into the periplasm. The accumulation of the expressed protein in the periplasm can be used for the purification of the protein and can avoid toxic effects that may occur when the protein resides in the periplasm.

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 332
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1180