Part:BBa_K243022

DsbA-Strep-DigA-Split Linker-Fok_a

This combination uses the benefits of an Streptavidin-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_a.The DsbA tag is used to transfer the produced protein into the periplasm.

Usage and Biology

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 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_a is more efficiently than the use of a combination of DigA tagged oligos with a construct containing Fok_i. To avoid interactions between the DigA-tag with the connected protein domain Fok_a 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_a to guarantee the independent function of both parts. this Linker is an improved part ove the optimal distance between the parts. It is important that the used linker has a certain flexibility and is long enough to avoid sterical interferences between the parts. If the linker is too long it might cause a 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 338
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1186