Coding

Part:BBa_K863020

Designed by: Isabel Huber   Group: iGEM12_Bielefeld-Germany   (2012-09-19)

bhal laccase from Bacillus halodurans with T7 promoter, RBS and His-tag

bhal laccase from Bacillus halodurans with T7 and HIS

Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 190
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


We aimed to express this protein in E. coli to study the laccase activity on synthetic substrates at different pH levels. E. coli BL21Star cells were transformed with these parts, each of which contained a T7 promoter and termination sequence flanking the coding sequences. Expression was induced with IPTG and protein expression was performed, after which cells were harvested followed by sonication in protein extraction buffer. A pET160-GW/CAT construct expressing the chloramphenicol acetyltransferase (CAT) protein was used as a positive control for induction. The soluble fraction was analyzed on a 12% SDS-PAGE gel followed by Coomassie staining (Fig. 1). We observed expression of the positive control CAT protein (~32 kDa), but no evidence of tthl or bhal expression (Fig 2.)


Fig 1. SDS PAGE of laccase proteins.

Nevertheless, we assumed there would be some functional laccase present, and proceeded with decolorisation assays.

We tested the laccases on the decolorization of bromophenol blue. We first experimented with a standard solution of laccase purified from Trametes versicolor by incubating bromophenol blue in a buffer containing copper sulphate with various quantities of Trametes laccase.

Next, we assayed the E. coli protein extracts for laccase activity at different pH levels. The positive control, from Trametes, worked very well as a positive control, but only at low pH. Aside from that, there was no significant difference between the absorbance of the laccases and the negative control (Fig 3.).




Fig 3. Absorbance values of laccase proteins (Trametes = Positive control; CAT = Negatice control)

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Categories
//cds/enzyme
//function/degradation
//proteindomain/degradation
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