Difference between revisions of "Part:BBa K3859014"

 
 
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The sfGFP is used in our construction of yeast barcode plasmid(fig.15A). sfGFP is a robustly folded green fluorescent protein which folds when even when fused to poorly folded polypeptides[1].
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<b><font size="+1.2"> Characterization </font></b>
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We designed a plasmid in Ecoil which contains sfGFP cassette first,  then replaced the sfGFP with our barcode using golden gate assembly. Next we cultured the E.coli with the edited plasmid on a LB agar plate and grew overnight at 37°C. Colonies that show no fluorescence contains the plasmids that have been successfully edited(fig.15B). The sfGFP cassette is used for us to estimate whether the barcodes were successfully transfered into plasmids.
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<div style='text-align: center;'>
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[[File:T--GreatBay_SZ--part_K3859014_15A.png|600px|thumb|center]]
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<b> 【fig.15A】LB plate grows with E.coil which contain sfGFP(before golden gate assembly) </b>
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[[File:T--GreatBay_SZ--part_K3859014_15B.png|600px|thumb|center]]
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<b> 【fig.15B】LB plate grows with E.coil which does not contain sfGFP(after golden gate assembly) </b>
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<b><font size="+1.2"> Reference </font></b>
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1. Pedelacq, J. D., & Cabantous, S. (2019). Development and Applications of Superfolder and Split Fluorescent Protein Detection Systems in Biology. International journal of molecular sciences, 20(14), 3479. https://doi.org/10.3390/ijms20143479
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Latest revision as of 13:09, 21 October 2021


Ura3 upstream homologous region-Promoter-sfGFP-terminator-Ura3 downstream homologous region

The sfGFP is used in our construction of yeast barcode plasmid(fig.15A). sfGFP is a robustly folded green fluorescent protein which folds when even when fused to poorly folded polypeptides[1].

Characterization

We designed a plasmid in Ecoil which contains sfGFP cassette first, then replaced the sfGFP with our barcode using golden gate assembly. Next we cultured the E.coli with the edited plasmid on a LB agar plate and grew overnight at 37°C. Colonies that show no fluorescence contains the plasmids that have been successfully edited(fig.15B). The sfGFP cassette is used for us to estimate whether the barcodes were successfully transfered into plasmids.

T--GreatBay SZ--part K3859014 15A.png

【fig.15A】LB plate grows with E.coil which contain sfGFP(before golden gate assembly)

T--GreatBay SZ--part K3859014 15B.png

【fig.15B】LB plate grows with E.coil which does not contain sfGFP(after golden gate assembly)

Reference

1. Pedelacq, J. D., & Cabantous, S. (2019). Development and Applications of Superfolder and Split Fluorescent Protein Detection Systems in Biology. International journal of molecular sciences, 20(14), 3479. https://doi.org/10.3390/ijms20143479


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NotI site found at 3
    Illegal NotI site found at 3523
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1842
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1731
    Illegal BsaI.rc site found at 713
    Illegal SapI.rc site found at 897
    Illegal SapI.rc site found at 2628