Difference between revisions of "Part:BBa K243025:Design"

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===Design Notes===
 
===Design Notes===
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The linker length and the choice of the purification tag in combination with the anticalin tag allowed us several combination possibilities, which we had to prove to achieve the best combination.
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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 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, whereas the combination of DigA tagged oligo and constructs consisting Fok_a seems to be more efficient. To avoid interactions between the FluA tag with the connected protein domain Fok_i 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 a instability of the whole construct.
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[https://static.igem.org/mediawiki/parts/d/dd/Freiburg09_Strep-FluA-ML-Fok_is.txt Commented GenBank file]
 
[https://static.igem.org/mediawiki/parts/d/dd/Freiburg09_Strep-FluA-ML-Fok_is.txt Commented GenBank file]
  

Latest revision as of 01:57, 22 October 2009

Strep-FluA-Middle Linker-Fok_i


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 278
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

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 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, whereas the combination of DigA tagged oligo and constructs consisting Fok_a seems to be more efficient. To avoid interactions between the FluA tag with the connected protein domain Fok_i 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 a instability of the whole construct. Commented GenBank file

Source

Combined by serial clonig steps.

References