Composite

Part:BBa_K1688005

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

dTomato and ModLac with HlyA tag (inc RBS, NahR/Psal promoter system) Sequence and Features

Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 786
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 77
    Illegal NgoMIV site found at 618
    Illegal NgoMIV site found at 2277
  • 1000
    COMPATIBLE WITH RFC[1000]

BBa_J61051, nahR with nahR and sal promoters (Pr and Psal), codes for the inducible repressor NahR, which suppresses Psal in the absence of salicylate. This biobrick makes it possible to regulate the expression of the red fluorescent protein, dTomato, and the modified D439A/M510L CueO laccase (ModLac) with attached N-His6 tag and C-export tag (HlyA, BBa_K554002).

Usage and Biology

The dTomato protein is a fluorescent protein dimer, created by direct evolution of the wild-type DsRed, from Discosoma sp. (Shaner et al, - Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein, 2004). The dTomato protein is a fluorescent dimer protein that emits orange-red light when it is excited by green-yellow light. It is preferable to use – especially in self-made fluorometry tests – because the excitation wavelengths and the emission wavelengths don't overlap as much as in other fluorescent proteins. The dTomato excitation peak is at 554 nm and 50% of it is at 510 nm. Also, its emission peak is at 581 nm and its 50% emission at 629 nm.

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.




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