Part:BBa_K2037003

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.