Difference between revisions of "Part:BBa K2940002"
 
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<partinfo>BBa_K2940002 short</partinfo>
 
<partinfo>BBa_K2940002 short</partinfo>
  
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Improved Bpul (Laccase from <I>Bacillus pumilus</I>
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'''Improvement of K863001'''
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Bpul (Laccase from <I>Bacillus pumilus</I>) with improved enzymatic activity. Generated by error prone PCR mediated directed evolution.
  
 
===Usage and Biology===
 
===Usage and Biology===
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'''Characterization ABTS assay'''
 
'''Characterization ABTS assay'''
  
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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.
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After mutant library generation vie error prone PCR. 92 mutant laccase enzymes were prescreened via a plate based dye degradation assay. This assay showed 23 which produced functional Bpul laccase enzymes. Enzyme activity for the mutants was assessed by ABTS assay, four of which displayed higher activity when compared to wild type Bpul (Shown in the graph). One (M26) was shown to have a 0.8 fold increase in activity when compared to wild type Bpul. Sequencing showed eight point mutations present in M26 when compared to wild type Bpul.  
  
  
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[[Image:T--Edinburgh OG--FigureScreening_Nathan.png|400px|]]
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[[Image:T--Edinburgh OG--FigureScreening_Nathan.png|thumb|92 randomly selected mutants were prescreened on agarplates containing 25 μM trypan blue. Transformed colonies were picked and placed onto dye plate followed by incubation at 37 C for 48 h. Yellow squaresindicate selected colonies based on dye degradation. The blue squareindicates WT Bpul. The red square indicates pET28a-GG-RFP-CD.]]
  
  
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'''Results'''
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[[Image:T--Edinburgh OG--FigureBpul2 Nathan.png|thumb|Mutants with activity higher than WT Bpul as determined byABTS assay are shown. The results shown are mean ̆standard error forthree replicates. Significant differences between wild type (WT) Bpul and mutants P<0.05 (**)]]
  
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Laccase activity was determine from Bpul.
 
  
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[[Image:T--Edinburgh OG--FigureBpul2 Nathan.png|200px|]]
 
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<span class='h3bb'>Sequence and Features</span>
 
 
<partinfo>BBa_K2940002 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2940002 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 01:02, 22 October 2019


Improved Bpul

Improvement of K863001

Bpul (Laccase from Bacillus pumilus) with improved enzymatic activity. Generated by error prone PCR mediated directed evolution.

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

After mutant library generation vie error prone PCR. 92 mutant laccase enzymes were prescreened via a plate based dye degradation assay. This assay showed 23 which produced functional Bpul laccase enzymes. Enzyme activity for the mutants was assessed by ABTS assay, four of which displayed higher activity when compared to wild type Bpul (Shown in the graph). One (M26) was shown to have a 0.8 fold increase in activity when compared to wild type Bpul. Sequencing showed eight point mutations present in M26 when compared to wild type Bpul.


92 randomly selected mutants were prescreened on agarplates containing 25 μM trypan blue. Transformed colonies were picked and placed onto dye plate followed by incubation at 37 C for 48 h. Yellow squaresindicate selected colonies based on dye degradation. The blue squareindicates WT Bpul. The red square indicates pET28a-GG-RFP-CD.


Mutants with activity higher than WT Bpul as determined byABTS assay are shown. The results shown are mean ̆standard error forthree replicates. Significant differences between wild type (WT) Bpul and mutants P<0.05 (**)



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]