Generator

Part:BBa_K1216001

Designed by: Parvathi Chandran   Group: iGEM13_ETH_Zurich   (2013-08-29)
Revision as of 14:24, 4 October 2013 by Niederbm (Talk | contribs)

Alkaline Phosphatase (phoA) from Citrobacter

The alkaline phosphatase is a periplasmic homodimeric hydrolase. Each monomer contains 429 amino acids.
3D representation of the alkaline phosphatase from [http://www.rcsb.org/pdb/explore/explore.do?structureId=1ANJ RCSB]

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

Usage and Biology

Alkaline phosphatases are used as reporter enzymes in different assays such as Western Blotting and in situ hybridization[1]. Testing human blood for Alkaline Phosphatase levels is a routine test that can reveal different conditions[2].

Alkaline phosphatases cleave phosphate groups from organic compounds by hydrolysis while retaining stereochemistry[3]. A good explanation of the mechanism can be found [http://chemwiki.ucdavis.edu/Organic_Chemistry/Organic_Chemistry_With_a_Biological_Emphasis/Chapter_10%3A_Phosphoryl_transfer_reactions/Section_10.3%3A_Hydrolysis_of__phosphates here].
Alkaline phosphatases, respectively their serum levels, are also related to several diseases e.g. metabolic myopathies and Paget Disease. [4]

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 340
    Illegal NgoMIV site found at 787
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterization

[http://2013.igem.org/Team:ETH_Zurich ETH Zurich 2013] used PhoA 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 PhoA with 4-MU-phosphate.

References

  1. Molecular Cell Biology, Fifth Edition, W.H. Freeman & Co., 2004.
  2. [http://www.nlm.nih.gov/medlineplus/ency/article/003470.htm Medline Plus]
  3. [http://chemwiki.ucdavis.edu/Organic_Chemistry/Organic_Chemistry_With_a_Biological_Emphasis/Chapter_10%3A_Phosphoryl_transfer_reactions/Section_10.3%3A_Hydrolysis_of__phosphates Section 10.3: Hydrolysis of phosphates]
  4. Adams & Victor's Principles Of Neurology, 7th edition, McGraw-Hill Professional, 2000.
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