Coding

Part:BBa_K3279008

Designed by: Liang Siwen   Group: iGEM19_CAU_China   (2019-10-15)
Revision as of 15:54, 15 October 2019 by SvenL (Talk | contribs)


cenA gene fused with INP-N sequence

This part is a improvement of the previous part cenA gene (BBa_K118023). With the fusion with INP-N, the endoglucanase coded by cenA gene can rivet on the surface and achieve E.coli surface display[1].

Usage and Biology

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 333
    Illegal BamHI site found at 733
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 75
    Illegal NgoMIV site found at 408
    Illegal AgeI site found at 1051
    Illegal AgeI site found at 1414
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterization from iGEM19_CAU_China

This part is a improvement of the previous part cenA gene (BBa_K118023). With the fusion with INP-N, the endoglucanase coded by cenA gene can rivete on the surface and achieve E.coli surface display. We linked this part into a pET30a(+) backbone, then transformed this plasmid into BL21. We induced this recombinant E.coli strain overnight under the condition of 16℃ 0.08 mM. See Fig.1[1].

To confirm whether it worked or not, we first detected the presence of the target protein by immunofluorescence staining and compared the fluorescence pattern and compared it with a negative control where cellulases was not fused with INP. We attached the His-tag to the fusion protein, so that it could allow an anti-His-tag primary antibody to combine the fusion protein and then let a fluorescence secondary antibody recognize them. As the result, we could spot the fluorescence of GFP when INP-N was fused with cellulase. See Fig.2[1].

Then the effect of fusion on enzyme activity was detected by measuring the cellulose degradation ability using CMC-Na as substrate. See Fig.3[1].


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Categories
Parameters
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