Difference between revisions of "Part:BBa K2037003"

 
 
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<partinfo>BBa_K2037003 parameters</partinfo>
 
<partinfo>BBa_K2037003 parameters</partinfo>
 
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We synthesized ‘EucgrG03098’ from Eucalyptus to assess its ability to reduce oxygen at the PBEC graphene cathode. An SP6 phage promoter (BBa_K2037000) was annealed to the basic part for in vitro expression of the laccase (Promega TNT SP6 Coupled Wheatgerm Extract System).
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A bromophenol blue assay was performed together with an existing laccase (BBa_K863020) for comparison, but neither caused a decolourisation of the substrate (Fig. 1).
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A positive control was included in the form of a standard solution of laccase purified from Trametes versicolor. The laccase was incubated in bromophenol blue in a buffer containing copper sulphate with various quantities of Trametes laccase. At least 20mU is necessary for sufficient decolourisation (Fig. 2).
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<html><img src="https://static.igem.org/mediawiki/parts/a/a3/T--Pretoria_UP--In_vitro_expression.png" height="300"></html>
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Fig.1: Bromophenol blue decolourisation assay of two different laccase parts.
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<html><img src="https://static.igem.org/mediawiki/parts/d/d7/T--Pretoria_UP--Laccase_positive_control.png" height="300"></html>
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Fig. 2: Bromophenol blue assay of Trametes versicolor derived laccase.
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The results can be seen as inconclusive, since the SP6 promoter could not be verified as a working part. This means that the laccase gene might be functioning, but it was not expressed in sufficient amounts due to a weak promoter. Another explanation might be that the in vitro expression system used in this process did not yield a high concentration of the laccase, which lead to no decolourisation.

Latest revision as of 03:47, 30 October 2016


Laccase from Eucalyptus grandis

Laccase 'EucgrG03098' protein from Eucalyptus grandis with His (x6) tag after start codon.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 499


We synthesized ‘EucgrG03098’ from Eucalyptus to assess its ability to reduce oxygen at the PBEC graphene cathode. An SP6 phage promoter (BBa_K2037000) was annealed to the basic part for in vitro expression of the laccase (Promega TNT SP6 Coupled Wheatgerm Extract System). A bromophenol blue assay was performed together with an existing laccase (BBa_K863020) for comparison, but neither caused a decolourisation of the substrate (Fig. 1). A positive control was included in the form of a standard solution of laccase purified from Trametes versicolor. The laccase was incubated in bromophenol blue in a buffer containing copper sulphate with various quantities of Trametes laccase. At least 20mU is necessary for sufficient decolourisation (Fig. 2).



Fig.1: Bromophenol blue decolourisation assay of two different laccase parts.




Fig. 2: Bromophenol blue assay of Trametes versicolor derived laccase.

The results can be seen as inconclusive, since the SP6 promoter could not be verified as a working part. This means that the laccase gene might be functioning, but it was not expressed in sufficient amounts due to a weak promoter. Another explanation might be that the in vitro expression system used in this process did not yield a high concentration of the laccase, which lead to no decolourisation.