DNA

Part:BBa_K2940002

Designed by: Nathan Wu   Group: iGEM19_Edinburgh_OG   (2019-10-15)
Revision as of 02:33, 21 October 2019 by Ivanjaubert (Talk | contribs) (→‎Characterization)


Improved Bpul

Improved Bpul (bacterial laccase Bpul from Bacillus pumilus)

Usage and Biology

Laccases are enzymes with the potential to biodegrade synthetic dyes with different chemical structures. These enzymes are able to oxidize a wide range of phenolic substrates without the presence of additional co-factors. This improved Bpul enzyme has a major bioactivity for degrading azo dyes.


Characterization

Libraries of laccase mutants were generated using error prone Taq PCR (New England BioLabs). The mutant libraries of assembled PelB and Bpul were then cloned into pET28a-GG-RFP-CD via Golden Gate cloning for expression.

Characterization ABTS assay

98 mutant laccase enzymes were assayed for enzymatic activity using ABTS assay. This assay showed four mutants with improved activity (one with a significant increase). This mutant was sequenced and showed eight-point mutations when compared to WT Bpul.


T--Edinburgh OG--FigureScreening Nathan.png


Results


T--Edinburgh OG--FigureBpul2 Nathan.png

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 726
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 123
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 726
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 726
    Illegal NgoMIV site found at 490
    Illegal NgoMIV site found at 958
    Illegal AgeI site found at 246
    Illegal AgeI site found at 1123
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None