Difference between revisions of "Part:BBa K243017"

Revision as of 21:04, 21 October 2009

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_a is more efficiently than the use of a combination of FluA tagged oligo with a construct containing Fok_a. To avoid interactions between the DigA-tag with the connected protein domain Fok_a we applied the split Linker. This Linker is an improved part of the team Freiburg08 and it is a suitable linker for fusion proteins. The properties of the split linker are an good compromise between the stability and the distance for the connection between protein domain Fok_a and the anticalin tag.

Sequence and Features