Generator

Part:BBa_K1216000

Designed by: Angela Aeschbach   Group: iGEM13_ETH_Zurich   (2013-08-29)
Revision as of 14:22, 4 October 2013 by Niederbm (Talk | contribs) (→‎Characterization)

β-Glucuronidase (gusA) from Bacillis Subtilis

gusA (also called uidA[1]) encodes β-Glucuronidase, an intracellular enzyme that catalyzes the hydrolysis of β-D-glucuronides.
3D representation of the β-Glucuronidase from [http://www.rcsb.org/pdb/explore/explore.do?structureId=3K46 RCSB]


A form of this protein with added TEV and poly-HIS tags can be found here.

Usage and Biology

β-Glucuronidase is used as a fusion protein marker in higher plants, due to them lacking intrinsic β-Glucuronidase activity[2]. Generally it can be used as reporter enzyme with detection by biochemical activity assays, immunological assays or by histochemical staining of tissue sections or cells[3].


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 538
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterization

[http://2013.igem.org/Team:ETH_Zurich ETH Zurich 2013] used GusA in their project as reporter enzyme. To make predictions about their system, the model obviously needed parameters, so they decided to conduct fluorometric assays in order to obtain Km values.To this end bacterial cells were grown until in exponential growth phase. Upon reaching this, gene expression was induced by AHL (see [http://http://2013.igem.org/Team:ETH_Zurich/Infoproc their system]). After another 4-5h of growth, cells were harvested and lysed, the cell free extract used for the fluorometric assay. The properly diluted CFX was measured on a 96 well plate in triplicates per substrate concentration. A plate reader took measurements at λEx. 365nm and ΛEm. 445nm. The obtained data was evaluated and finally fitted to Michaelis-Menten-Kinetics with SigmaPlot™. See the resulting graph below.

Michaelis-Menten-Kinetics of GusA with 4-MU-β-D-glucuronide.

References

  1. [http://ecoliwiki.net/colipedia/index.php/uidA:Gene ecoliwiki]
  2. Jefferson A R, "GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants.", EMBO J. 1987 December 20; 6(13): 3901–3907 [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC553867/]
  3. [http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Bulletin/gusabul.Par.0001.File.tmp/gusabul.pdf Sigma Aldrich]
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