Difference between revisions of "Part:BBa K243010:Design"

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===Design Notes===
 
===Design Notes===
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The choice of the linker, lipocalin tag and purification tag allows us serval different combinations.
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The cloning steps were planned theoretically before we started the work in the wet lab.
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The combination of HisTag linked with a FluA tag connected by the Split Linker approved as a good way to express the [https://parts.igem.org/wiki/index.php?title=Part:BBa_K243001 inactive protein domain] of our universal endonuclease.<br><br>
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[[Image:Freiburg09 Hisfluasplfoki.jpg|550x350px]]<br>
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<p class=MsoNormal><b>part of universal restriction enzyme.</b> Blue:
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DNA strand; Red: 16bp long Oligos, tag as indicated in the picture.<br> Ochre:
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Fluorescein A binding lipocalin;light Blue: inactive FokI cleavage domain;</p> 
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We used the [https://parts.igem.org/Part:BBa_K157009 split linker] because it is an improved part of the team [http://2008.igem.org/Team:Freiburg Freiburg08] and a suitable linker for fusion proteins. The properties of the split linker are a good compromise between the stability and the distance for the connection between protein domain Fok_a and the anticaline tag.The dimerization partner to this part needs another combination of anticaline and the protein domain Fok_a, because the labeled oligonucleotides had different anticalins.
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The parts are fused according to [https://parts.igem.org/Assembly_standard_25 RFC 25].<br>
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[https://static.igem.org/mediawiki/parts/6/6a/HisFluASplitFoki.txt Commented GenBank file]
  
 
===Source===
 
===Source===

Latest revision as of 03:09, 22 October 2009

His-FluA-Split 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

The cloning steps were planned theoretically before we started the work in the wet lab. The combination of HisTag linked with a FluA tag connected by the Split Linker approved as a good way to express the inactive protein domain of our universal endonuclease.

Freiburg09 Hisfluasplfoki.jpg

part of universal restriction enzyme. Blue: DNA strand; Red: 16bp long Oligos, tag as indicated in the picture.
Ochre: Fluorescein A binding lipocalin;light Blue: inactive FokI cleavage domain;

We used the split linker because it is an improved part of the team [http://2008.igem.org/Team:Freiburg Freiburg08] and a suitable linker for fusion proteins. The properties of the split linker are a good compromise between the stability and the distance for the connection between protein domain Fok_a and the anticaline tag.The dimerization partner to this part needs another combination of anticaline and the protein domain Fok_a, because the labeled oligonucleotides had different anticalins. The parts are fused according to RFC 25.
Commented GenBank file

Source

Combined the parts by serial cloning steps.

References