Difference between revisions of "Part:BBa K243016:Design"

(→‎Design Notes)
(→‎Design Notes)
 
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===Design Notes===
 
===Design Notes===
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The choice of the linker, lipocalin tag and purification tag allows us several different combinations.
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We applied the His tag to enable a simultaneous purification of constructs with a Strep tag. 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/b/bc/Freiburg09_His-FluA-LL-Fok_is.txt Commented GenBank file]
 
[https://static.igem.org/mediawiki/parts/b/bc/Freiburg09_His-FluA-LL-Fok_is.txt Commented GenBank file]
  

Latest revision as of 02:13, 22 October 2009

His-FluA-Long 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 272
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

We applied the His tag to enable a simultaneous purification of constructs with a Strep tag. 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.

Commented GenBank file

Source

Combined the parts by serial cloning steps.

References