Difference between revisions of "Part:BBa K243023:Design"
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===Design Notes=== | ===Design Notes=== | ||
The linker length and the choice of the purification tag in combination with the anticalin tag allowed us several combination possiblities, which we had to prove to achieve the best combiantion. | The linker length and the choice of the purification tag in combination with the anticalin tag allowed us several combination possiblities, which we had to prove to achieve the best combiantion. | ||
| + | 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. FoIn this case the purification with Strep tag is more specific. The used FluoresceinA tag allows the measurement by quenching and the coupling to a fluorescein linked oligo. Emanating from our 3D modeling, this combination of FluA tagged oligo and the construct containing the protein domain Fok_i is more efficient than the use of a combination of DigA tagged oligo with a construct containing Fok_i. To avoid interactions between the FluA tag with the connected protein domain Fok_i we applied the Long Linker. The Linker itself has no influence on the connected parts. We decided to use the Long Linker for this construct to get a longer distance between FluA tag and Fok_i to guarantee the independent function of both parts and also 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 sterical interferences between the parts. If the linker is too long it might cause the instability of the whole construct. | ||
[https://static.igem.org/mediawiki/parts/2/27/Freiburg09_Strep-FluA-LL-Fok_is.txt Commented GenBank file] | [https://static.igem.org/mediawiki/parts/2/27/Freiburg09_Strep-FluA-LL-Fok_is.txt Commented GenBank file] | ||
Latest revision as of 02:05, 22 October 2009
Strep-FluA-Long Linker-Fok_i
- 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]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
The linker length and the choice of the purification tag in combination with the anticalin tag allowed us several combination possiblities, which we had to prove to achieve the best combiantion. 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. FoIn this case the purification with Strep tag is more specific. The used FluoresceinA tag allows the measurement by quenching and the coupling to a fluorescein linked oligo. Emanating from our 3D modeling, this combination of FluA tagged oligo and the construct containing the protein domain Fok_i is more efficient than the use of a combination of DigA tagged oligo with a construct containing Fok_i. To avoid interactions between the FluA tag with the connected protein domain Fok_i we applied the Long Linker. The Linker itself has no influence on the connected parts. We decided to use the Long Linker for this construct to get a longer distance between FluA tag and Fok_i to guarantee the independent function of both parts and also 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 sterical interferences between the parts. If the linker is too long it might cause the instability of the whole construct.
Source
Combined by serial clonig steps.