Generator

Part:BBa_K1688007

Designed by: Fredrik Lindeberg   Group: iGEM15_Uppsala   (2015-09-15)

ModLac laccase with His-tag and HlyA export tag (inc RBS and J23110 promoter) Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 310
  • 1000
    COMPATIBLE WITH RFC[1000]

Assembly of Anderson promoter BBa_J23110 and RBS + ModLac with HlyA tag (BBa_K1688006). ModLac is 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.

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 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.

Design Notes

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 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.

References

Kataoka, K, Kogi H, Tsujimura S, Sakurai T. 2012. Modifications of laccase activities of copper efflux oxidase, CueO by synergistic mutations in the first and second coordination spheres of the type I copper center. Faculty of pure and applied science , University of Tsukuba,


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n/aModLac laccase with His-tag and HlyA export tag (inc RBS and J23110 promoter)