Difference between revisions of "Part:BBa K243021"

Revision as of 21:16, 21 October 2009

DsbA-His-DigA-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. The DsbA tag is used to transfer the produced protein into the periplasm.

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 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 DigoxigeninA-tag allows the 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 a suitable linker 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. The additional linkage of the construct wit DsbA allows the export of the protein into the periplasm. The accumulation of the expressed protein in the periplasm can be used for the purification of the protein.

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 332
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 1180

@@ Line 3: / Line 3: @@
-This combination uses the benefits of an His-tag (Polyhistidin-tag) for purification. It is also linked with a DigoxigenineA-tag       [https://parts.igem.org/wiki/index.php?title=Part:BBa_K243003 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.The DsbA tag is used to transfer the produced protein into the periplasm.
+This combination uses the benefits of a His-tag (Polyhistidin-tag) for purification. It is also linked with a DigoxigeninA tag       [https://parts.igem.org/wiki/index.php?title=Part:BBa_K243003 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. The DsbA tag is used to transfer the produced protein into the periplasm.
@@ Line 9: / Line 9: @@
-This composite part is one part of our universal endonuclease and it needs another composite part like BBa_K243010 to built to a functional heterodimer. The DigA tag leads the part to DNA which is hybridized with an Digoxigenin labeled oligonucleotide. The split linker makes a distance from 51bp between the DigA and the linked Fok_a protein domain. The His-Tag serve as purification tag for Ni-NTA column purification.
+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 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 DigoxigeninA-tag allows the coupling to an flurescein linked oligo. Emanating from our 3D modeling this combination of DigA tagged oligos and the construct containing the protein domain Fok_a is more efficiently 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 of 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 a suitable linker for fusion proteins. The properties of the split linker are an good compromise between the stability and the distance for the connection between protein domain Fok_a and the anticalin tag. The additional linkage of the construct wit DsbA allows the export of the protein into the periplasm. The accumulation of the expressed protein in the periplasm can be used for the purification of the protein.
+We applied the His tag to enable a simultaneous purification of constructs with a Strep tag. The used DigoxigeninA-tag allows the 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 a suitable linker 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. The additional linkage of the construct wit DsbA allows the export of the protein into the periplasm. The accumulation of the expressed protein in the periplasm can be used for the purification of the protein.
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