Difference between revisions of "Part:BBa K243024"
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<partinfo>BBa_K243024 short</partinfo> | <partinfo>BBa_K243024 short</partinfo> | ||
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| − | the independent function of FluA tag and the protein domain Fok_a | + | This combination uses the benefits of a Streptavidin tag for purification. It is also linked with a FluoresceinA tag. The Middle Linker (GlySerGlyGly)x2 connects the parts and adds additional space between them to guarantee the independent function of FluA tag and the protein domain Fok_a.<br> |
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===Usage and Biology=== | ===Usage and Biology=== | ||
| + | This composite part is one part of our universal endonuclease and it needs another composite part like BBa_K243012 to build a functional heterodimer. The FluA tag guides the part to DNA which is hybridized with a Fluorescein labeled oligonucleotide. The middle linker creates a distance of 24bp between the FluA and the linked Fok_a protein domain. The StrepTag serves as purification tag for Streptavidin column purification. | ||
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| + | We applied the Streptavidine tag to enable a simultaneous purification of constructs with a His tag. Strep tag also shows a higher affinity towards Strep-Tactin than His tag. In this case the purification with Strep tag is more specific. The used FluoresceinA tag allows the measurement by quenching and the coupling to a flurescein linked oligo. Emanating from our 3D modeling this combination of FluA tagged oligo and the construct containing the protein domain Fok_a is not as efficient as the use of a combination of DigA tagged oligo with a construct containing Fok_a. To avoid interactions between the FluA tag with the connected protein domain Fok_a we applied the Middle Linker. The Linker itself has no influence on the connected parts. We decided to use the Middle Linker for this construct to prove the optimal distance between the parts. It is important that the used linker has a certain flexibility and is long enough to avoid steric interferences between the parts. If the linker is too long it might cause the instability of the whole construct. | ||
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| + | Sequence and Features | ||
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
Latest revision as of 21:42, 21 October 2009
Strep-FluA-Middle Linker-Fok_a
This combination uses the benefits of a Streptavidin tag for purification. It is also linked with a FluoresceinA tag. The Middle Linker (GlySerGlyGly)x2 connects the parts and adds additional space between them to guarantee the independent function of FluA 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_K243012 to build a functional heterodimer. The FluA tag guides the part to DNA which is hybridized with a Fluorescein labeled oligonucleotide. The middle linker creates a distance of 24bp between the FluA and the linked Fok_a protein domain. The StrepTag serves as purification tag for Streptavidin column purification.
We applied the Streptavidine tag to enable a simultaneous purification of constructs with a His tag. Strep tag also shows a higher affinity towards Strep-Tactin than His tag. In this case the purification with Strep tag is more specific. The used FluoresceinA tag allows the measurement by quenching and the coupling to a flurescein linked oligo. Emanating from our 3D modeling this combination of FluA tagged oligo and the construct containing the protein domain Fok_a is not as efficient as the use of a combination of DigA tagged oligo with a construct containing Fok_a. To avoid interactions between the FluA tag with the connected protein domain Fok_a we applied the Middle Linker. The Linker itself has no influence on the connected parts. We decided to use the Middle Linker for this construct to prove the optimal distance between the parts. It is important that the used linker has a certain flexibility and is long enough to avoid steric interferences between the parts. If the linker is too long it might cause the instability of the whole construct.
Sequence and Features Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 278
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 1075