Difference between revisions of "Part:BBa K243010"

Latest revision as of 04:36, 22 October 2009

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 of fluorescein binding by quenching of the fluorescence signal.

Please visit the BioBrick entry for the functional Fok_a part for protein interaction and experimental details.

Usage and Biology

This composite part is one main part of our universal endonuclease and it needs another composite part such as BBa_K243017 BBa_K243017 to built to a functional heterodimer. The FluA anticalin targets the complex to DNA which is hybridized with a fluorescein-labeled oligonucleotide. The Split Linker creates a distance of 17 aa between the FluA and the Fok_i protein domain to ensure the formation of an active cleavage conformation. The HisTag serves as a purification tag for Ni-NTA columns.

Purification

We purified this part of our universal endonuclease utilizing a Ni-NTA column and proved the expression of the protein with a subsequent Western Blot.

Western Blot: His-FluA-SplitLi-Fok_i expressed using the pEx vector;
lanes: (1)NEB prestained protein marker broad range; (2)pooled elution fractions 2-5; (3)empty lane; (4-6) positive controls (pure His-tagged proteins)

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
COMPATIBLE WITH RFC[1000]

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K243010 short</partinfo>
-These part units some of our parts. The combination of a [https://parts.igem.org/wiki/index.php?title=Part:BBa_K157011 Histidin-Tag], a [https://parts.igem.org/wiki/index.php?title=Part:BBa_K157004 Anticalin-Tag] and the [https://parts.igem.org/wiki/index.php?title=Part:BBa_K157009 linked] protein domain Fok_a, makes it possible to purifier and detect the function of the protein. The [https://parts.igem.org/wiki/index.php?title=Part:BBa_K157004 fluoresceinA tag] allows the measurement by quenching and the coupling to an flurescein linked oligo.<br>
+In combination with the [https://parts.igem.org/wiki/index.php?title=Part:BBa_K243036 Fok_a composite part] this biobrick can be used to create an active universal restriction enzyme. The combination of a [https://parts.igem.org/wiki/index.php?title=Part:BBa_K157011 Polyhistidin-Tag], a [https://parts.igem.org/wiki/index.php?title=Part:BBa_K157004 flourescein-binding anticalin] and the [https://parts.igem.org/wiki/index.php?title=Part:BBa_K157009 linker] protein domain with Fok_a makes it possible to purify and detect the fusion protein. The [https://parts.igem.org/wiki/index.php?title=Part:BBa_K157004 fluoresceinA tag] allows the measurement of fluorescein binding by quenching of the fluorescence signal.<br><br>
+Please visit [https://parts.igem.org/wiki/index.php?title=Part:BBa_K243036 the BioBrick entry for the functional Fok_a part for protein interaction and experimental details].
+===Usage and Biology===
+This composite part is one main part of our universal endonuclease and it needs another composite part such as [https://parts.igem.org/wiki/index.php?title=Part:BBa_K243010 BBa_K243017 BBa_K243017] to built to a functional heterodimer. The FluA anticalin targets the complex to DNA which is hybridized with a fluorescein-labeled oligonucleotide. The Split Linker creates a distance of 17 aa between the FluA and the Fok_i protein domain to ensure the formation of an active cleavage conformation. The HisTag serves as a purification tag for Ni-NTA columns.
 ===Purification===
-We achieved to purify one part of our universal endonuclease by a Histidin column purification and proved it with a following Western Blot.
+We purified this part of our universal endonuclease utilizing a Ni-NTA column and proved the expression of the protein with a subsequent Western Blot.<br>
+<br>
 [[Image:WB09.10.02jpg.jpg]]<br>
-Western Blot: His-Flu_a-Split_Fok_i in pEx;<br> lanes: NEB prestained marker broad range,
+Western Blot: His-FluA-SplitLi-Fok_i expressed using the pEx vector;<br> lanes: (1)NEB prestained protein marker broad range; (2)pooled elution fractions 2-5; (3)empty lane; (4-6) positive controls (pure His-tagged proteins)
-<br> pool of elution fractions 2-5, empty lane, 3 positive controls
-<!-- Add more about the biology of this part here
-===Usage and Biology===
 <!-- -->
-<span class='h3bb'>Sequence and Features</span>
+<span class='h3bb'>'''Sequence and Features'''</span>
 <partinfo>BBa_K243010 SequenceAndFeatures</partinfo>