Difference between revisions of "Part:BBa K1216003"
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<partinfo>BBa_K1216003 parameters</partinfo>
 
<partinfo>BBa_K1216003 parameters</partinfo>
 
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==Characterization==
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[[File:NagZ_colored.png|frame|right|<b>Figure 1.  Cell lysate from <i>E.Coli</i> overexpressing NagZ after reacting with pNP-GlcNac.</b>]]
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[http://2013.igem.org/Team:ETH_Zurich ETH Zurich 2013] used NagZ in their project as reporter enzyme.
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To test the functionality of the enzyme, cell lysate of <i>E.Coli</i> overexpressing NagZ was incubated with GlcNac.
 +
[[File:nagz_color_reaction.png|thumb|center|396px|<b>Figure 2.  Enzymatic reaction of NagZ with GlcNac.</b>]]
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Cell lysate for the assay described below was tested for active enzyme in the same way, but with the fluorescent substrate 4-MU-N-acetyl-β-D-glucosaminide. Unforunately this did not work until the wiki freeze.
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[[File:nagz_fluorescent_reaction.png|frame|center|<b>Figure 4.  Enzymatic reaction of NagZ with 4-MU-N-acetyl-β-D-glucosaminide.</b>]]
 
===References===
 
===References===
 
# Cheng et al. (2000). Molecular Characterization of the b-N-Acetylglucosaminidase of Escherichia coli and Its Role in Cell Wall Recycling. ''Journal of Bacteriology''. 182: 4836-4840.
 
# Cheng et al. (2000). Molecular Characterization of the b-N-Acetylglucosaminidase of Escherichia coli and Its Role in Cell Wall Recycling. ''Journal of Bacteriology''. 182: 4836-4840.
 
# [http://www.inalcopharm.com/cart.php?m=product_detail&p=6 Inalcopharm datasheet]
 
# [http://www.inalcopharm.com/cart.php?m=product_detail&p=6 Inalcopharm datasheet]

Revision as of 20:19, 4 October 2013

beta-N-Acetylglucosaminidase (nagZ) from Escherichia Coli

nagZ encodes β-N-Acetylglucosaminidase, a cytoplasmatic hydrolase which is involved in the murein tripeptide recycling pathway of E.coli[1].
3D representation of the β-N-Acetylglucosaminidase from [http://www.rcsb.org/pdb/explore/explore.do?structureId=4GVG RCSB]


Usage and Biology

β-N-Acetylglucosaminidase can be used as a reporter enzyme in a histochemical reaction with substrates like 5-Bromo-4-Chloro-3-Indolyl-N-Acetyl-β-D-Glucosaminide, which will after hydrolysis release a blue chromophore[2].



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 25
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 76


Characterization

Figure 1. Cell lysate from E.Coli overexpressing NagZ after reacting with pNP-GlcNac.

[http://2013.igem.org/Team:ETH_Zurich ETH Zurich 2013] used NagZ in their project as reporter enzyme. To test the functionality of the enzyme, cell lysate of E.Coli overexpressing NagZ was incubated with GlcNac.

Figure 2. Enzymatic reaction of NagZ with GlcNac.

Cell lysate for the assay described below was tested for active enzyme in the same way, but with the fluorescent substrate 4-MU-N-acetyl-β-D-glucosaminide. Unforunately this did not work until the wiki freeze.

Figure 4. Enzymatic reaction of NagZ with 4-MU-N-acetyl-β-D-glucosaminide.

References

  1. Cheng et al. (2000). Molecular Characterization of the b-N-Acetylglucosaminidase of Escherichia coli and Its Role in Cell Wall Recycling. Journal of Bacteriology. 182: 4836-4840.
  2. [http://www.inalcopharm.com/cart.php?m=product_detail&p=6 Inalcopharm datasheet]