Part:BBa_K243036

His-Dig-Split Linker-Fok_a

This combination uses the benefits of a His tag (Polyhistidin tag) for purification. It is also linked with a DigoxigeninA 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.

Usage and Biology

This composite part is one part of our universal endonuclease and it needs another composite part like BBa_K243010 to build a functional heterodimer. The DigA tag guides the part to the DNA which is hybridized with a Digoxigenin labeled oligonucleotide. The Split Linker creates a distance of 51bp between the DigA and the linked Fok_a protein domain. The His Tag serves as a purification tag for Ni-NTA column purification.

Application

At least we succeeded to produce a working universal endonuclease with this part in combination with the part BBa_K243010, by cotransformation into E.coli(XL1blue). The part BBa_K243010 was cloned into our expression vector BBa_K243033 and this part was cloned into the vector pJS419 BBa_K243035. The cutting assay was conducted with cleared cell lysate and DNA hybridized with oligonucleotides labeled with fluorescence molecule(Cy3). The two proteins linked to the hybridized oligonucleotides and cut them at the chosen site. We proved the cutting event by gel electrophoresis and excitation of the fluorescence.

Gellanes:1.Marker(1kb ladder mix)2.Cell extract+sample1 3.Cell extract+sample2 4.control w/o cell extract.

Method
The pictures show our gel, on the left excited by wavelength of 353 nm and on the right side filtered with the cutoff filter of 580nm. The first lane shows the marker only visible on the right gel picture, the second and third lane show the products of the cutting event made by our universal endonuclease. The comparison with the fourth lane shows that the original oligonucleotides have been cut. In the fourth lane only the oligonucleotides were loaded. Detection of the Cy3

To excite the Cy3-tagged nucleotides, a laser with a wavelength of 353 nm was used. To cut off the excitation wavelength in the pictures, a cutoff filter of 580 nm was used. In another approach the SteREO Lumar.V12 from Zeiss was used to excite the Cy3-tagged oligonucleotides.

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 272
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 1126