Translational_Unit

Part:BBa_K1688006

Designed by: Fredrik Lindeberg   Group: iGEM15_Uppsala   (2015-09-15)
Revision as of 21:31, 18 September 2015 by AdrianSilberman (Talk | contribs)

A modified laccase (D439A/M510L CueO) with N-His6 tag attached to it via a linker sequence and an export tag (HlyA, BBa_K554002) attached to its C-terminus. ModLac laccase with His-tag and HlyA export tag (inc RBS)

Usage and Biology

Laccases (originally from Chinese lacquer tree sap) are multicopper oxidases, that are employed in various industries, where they take part in beer maturation, textile dyeing, and enzymatic biofuel cells. Due to their broad specificity and ability to oxidize aromatic compounds, their application in bioremediation is a topic under investigation. The laccase we chose is a modified laccase, CueO, a laccase from E. coli. The modified CueO laccase that we synthesized had a double mutation (D439A/M510L) that has been proven to increase the enzymatic activity (Kataoka K et al. 2012). A polyhistidine-tag was also added at the N-terminus so that it could be purified easily.

The aim with doing modifications of the laccase (CueO) was to improve the enzymatic activity of the protein. This particular Biobrick also has an export tag (HlyA) attatched to the C-terminus. The enzymatic activities was measured on the enzyme without the HlyA-tag, so the results regarding the enzyme kinetics assay is necessarirly not transferable to this specific Biobrick.

The enzymatic activities of the laccases was measured using ABTS. ABTS is a commonly used substrate when evaluating reaction kinetics of specific enzymes. Due to its reduction potential, it acts as an effective electron donor. Since we are working with laccases, which are multi copper oxidases, which oxidize substrates, ABTS is a suitable substrate. ABTS will donate electron to reduce molecular oxygen. The oxidized ABTS has a different absorption spectrum and the reaction can thus be observed in a spectrophotometer.

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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 267
  • 1000
    COMPATIBLE WITH RFC[1000]


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Parameters
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