Part:BBa_K243010
His-FluA-Split Linker-Fok_i
In combination with the Fok_a composite part this biobrick can be used to create an active universal restriction enzyme. The combination of a Polyhistidin-Tag, a flourescein-binding anticalin and the linker protein domain with Fok_a makes it possible to purify and detect the fusion protein. The fluoresceinA tag allows the measurement by quenching and the coupling to a fluorescein linked oligo.
Usage and Biology
This composite part is one part of our universal endonuclease and it needs another composite part like BBa_K243017 BBa_K243017 to built to a functional heterodimer. The FluA tag leads the part to DNA which is hybridized with a fluorescein labeled oligonucleotide. The Split Linker creates a distance of 51bp between the FluA and the linked Fok_i protein domain. The HisTag serves as a purification tag for Ni-NTA column purification.
Purification
We purified this part of our universal endonuclease by NiNTA column purification and proved the expression of the protein with a following Western Blot.
Western Blot: His-FluA-SplitLi-Fok_i in pEx;
lanes: NEB prestained marker broad range,
pool of elution fractions 2-5, empty lane, 3 positive controls
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 272
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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