Difference between revisions of "Part:BBa K243016"

Revision as of 00:32, 22 October 2009

His-FluA-Long Linker-Fok_i

This combination uses the benefits of an His-tag for purification. It is also linked with a FluroescineA-tag. The Long Linker (GlySerGlyGly)x3 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_K243022to built to a functional heterodimer. The FluA tag leads the part to DNA which is hybridized with an Fluorescein labeled oligonucleotide. The long linker makes a distance from 36bp between the FluA and the linked Fok_i protein domain. The His-Tag serve as purification tag for 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 efficiently 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 Long Linker. The Linker itself has no influence of the connected parts. We decided to use the Long Linker for this construct to get a longer distance between FluA-tag and Fok_i to guarantee the independent function of both parts and also to prove the optimal distance between the parts. It is important that the used linker has certain flexibility and is long enough to avoid sterical interferences between the parts. If the linker is too long it might cause a instability of the whole construct.

Sequence and Features