Difference between revisions of "Part:BBa K243036"

Latest revision as of 04:39, 22 October 2009

His-Dig-Split Linker-Fok_a

This biobrick serves as one part of the universal endonuclease and possesses the catalytically active FokI domain Fok_a. When the expressed fusion protein comes into proximity of its counterpart, a fusion protein with the Fok_i domain, an active heterodimer can form. A terminal His tag (Polyhistidin tag) allows easy purification. The lipocalin DigoxigeninA (DigA) targets the fusion protein to labeled molecules such as oligonucleotides. 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 needs another composite part like BBa_K243010 to build a heterodimer functional as a restriction enzyme. The DigA guides the part to the DNA which is hybridized with a Digoxigenin labeled oligonucleotide. The Split Linker creates a distance of 17 aa between the DigA and the linked Fok_a protein domain. The His Tag serves as a purification tag for Ni-NTA columns.

Application

We succeeded to create a working universal endonuclease with this part in combination with the part BBa_K243010 by cotransformation into E.coli(XL1blue). The part BBa_K243010 (the Fok_i fusion protein) was cloned into our expression vector BBa_K243033 whereas this part was cloned into the vector pJS419 BBa_K243035. A cutting assay was conducted with crude E. coli cell lysate and DNA hybridized with oligonucleotides labeled with the fluophore Cy3. The two Fok fusion proteins can form a heterodimer which can bind to hybridized oligonucleotides and subsequently cut target DNA. We proved the cutting event by gel electrophoresis and excitation of fluorescence.

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

Method
The pictures show our agarose gels, on the left excited by wavelength of 535 nm and filtered with a 580 nm cutoff filter. On the left picture, the daylight image has been superimposed. The first lane shows the marker (DNA bands slightly visible on the left gel, dye on the right gel), the second and third lane show the products of the cutting event catalyzed by our universal endonuclease. In the fourth lane only the oligonucleotides were loaded. Comparison with the fourth lane shows that the original oligonucleotides have been cut to smaller products. Detection of the Cy3

To excite the Cy3-tagged nucleotides, a laser with a wavelength of 535 nm was used. To block the excitation wavelength when recording the pictures, a cutoff filter of 580 nm was used. In an alternative approach a Zeiss SteREO Lumar.V12 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

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K243036 short</partinfo>
-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.
+This biobrick serves as one part of the universal endonuclease and possesses the catalytically active FokI domain Fok_a. When the expressed fusion protein comes into proximity of its counterpart, a fusion protein with the Fok_i domain, an active heterodimer can form.
+A terminal His tag (Polyhistidin tag) allows easy purification. The lipocalin DigoxigeninA (DigA) targets the fusion protein to labeled molecules such as oligonucleotides. 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.
+This composite part is one part of our universal endonuclease and needs another composite part like BBa_K243010 to build a heterodimer functional as a restriction enzyme. The DigA guides the part to the DNA which is hybridized with a Digoxigenin labeled oligonucleotide. The Split Linker creates a distance of 17 aa between the DigA and the linked Fok_a protein domain. The His Tag serves as a purification tag for Ni-NTA columns.
-We applied the His tag to enable a simultaneous purification of constructs with a Strep tag. The used DigoxigeninA tag allows coupling to a Fluorescein linked oligo. Emanating from our 3D modeling this combination of DigA tagged oligos and the construct containing the protein domain Fok_a is more efficient than the use of a combination of FluA tagged oligos 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. The linker itself has no influence on 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 of the team Freiburg08 and it is suitable for fusion proteins. The properties of the Split Linker are a good compromise between the stability and the distance of the connection between protein domain Fok_a and the anticalin tag.
 ===Application===
+We succeeded to create a working universal endonuclease with this part in combination with the part BBa_K243010 by cotransformation into ''E.coli''(XL1blue). The part BBa_K243010 (the Fok_i fusion protein) was cloned into our expression vector BBa_K243033 whereas this part was cloned into the vector pJS419 BBa_K243035.
+A cutting assay was conducted with crude E. coli cell lysate and DNA hybridized with oligonucleotides labeled with the fluophore Cy3. The two Fok fusion proteins can form a heterodimer which can bind to hybridized oligonucleotides and subsequently cut target DNA. We proved the cutting event by gel electrophoresis and excitation of fluorescence.
+<br><br>
+[[Image:Freiburg09_Fok_80mer.jpg|250x400px]]<br>
+Agarose gel lanes:1.Marker (1kb ladder mix) 2.Cell extract+sample1 3.Cell extract+sample2 4.control w/o cell extract.<br>
+<br>
+[https://static.igem.org/mediawiki/parts/a/ac/Fok_in_vitro_80mer_cutting_assay.pdf Method]<br>
+The pictures show our agarose gels, on the left excited by wavelength of 535 nm and filtered with a 580 nm cutoff filter. On the left picture, the daylight image has been superimposed. The first lane shows the marker (DNA bands slightly visible on the left gel, dye on the right gel), the second and third lane show the products of the cutting event catalyzed by our universal endonuclease. In the fourth lane only the oligonucleotides were loaded. Comparison with the fourth lane shows that the original oligonucleotides have been cut to smaller products.
+'''Detection of the Cy3<br>'''<br>
+To excite the Cy3-tagged nucleotides, a laser with a wavelength of 535 nm was used. To block the excitation wavelength when recording the pictures, a cutoff filter of 580 nm was used. In an alternative approach a Zeiss SteREO Lumar.V12 was used to excite the Cy3-tagged oligonucleotides.
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