Difference between revisions of "Part:BBa K1216003"

(→‎Characterization)
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<b>The final construct was sequenced.</b>
 
<b>The final construct was sequenced.</b>
  
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[[File:NagZ_colored.png|frame|right|<b>Figure 1.  Liquid culture from <i>E.Coli</i> overexpressing NagZ after reacting with pNP-GlcNac.</b>]]
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[[File:NagZ3substrates.JPG|thumb|right|300px|<b>Figure 1.  Liquid cultures from <i>E.Coli</i> overexpressing NagZ after reacting with pNP-GlcNac, magenta-GlcNAc and X-GlcNAc.</b> The negative control shows a liquid culture without substrate added.]]
 
[http://2013.igem.org/Team:ETH_Zurich ETH Zurich 2013] used NagZ in their project as reporter enzyme.
 
[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, a liquid culture of <i>E.Coli</i> overexpressing NagZ was incubated with pNP-GlcNac.  
+
To test the functionality of the enzyme, a liquid culture of <i>E.Coli</i> overexpressing NagZ was incubated with its chromogenic substrates magenta-GlcNac (produces magenta color), pNP-GlcNac (produces yellow color) and X-GlcNac (produces blue color).  
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[[File:PNP-GluNAc.png|thumb|center|396px|<b>Figure 2.  Enzymatic reaction of NagZ with pNP-GlcNac.</b>]]
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[[File:NagZandSubstrates.png|thumb|center|470px|<b>Figure 2.  Enzymatic reaction of NagZ with X-GlcNAc (1), magenta-GlcNAc (2) or pNP-GlcNac (3).</b>]]
 
Cell lysate would have been tested for active enzyme in the same way, but with the fluorescent substrate 4-MU-N-acetyl-β-D-glucosaminide. Unfortunately, this did not work until the wiki freeze.
 
Cell lysate would have been tested for active enzyme in the same way, but with the fluorescent substrate 4-MU-N-acetyl-β-D-glucosaminide. Unfortunately, this did not work until the wiki freeze.
 
[[File:nagz_fluorescent_reaction.png|frame|center|<b>Figure 4.  Enzymatic reaction of NagZ with 4-MU-N-acetyl-β-D-glucosaminide.</b>]]
 
[[File:nagz_fluorescent_reaction.png|frame|center|<b>Figure 4.  Enzymatic reaction of NagZ with 4-MU-N-acetyl-β-D-glucosaminide.</b>]]

Revision as of 01:46, 28 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

The final construct was sequenced.

Figure 1. Liquid cultures from E.Coli overexpressing NagZ after reacting with pNP-GlcNac, magenta-GlcNAc and X-GlcNAc. The negative control shows a liquid culture without substrate added.

[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, a liquid culture of E.Coli overexpressing NagZ was incubated with its chromogenic substrates magenta-GlcNac (produces magenta color), pNP-GlcNac (produces yellow color) and X-GlcNac (produces blue color).

Figure 2. Enzymatic reaction of NagZ with X-GlcNAc (1), magenta-GlcNAc (2) or pNP-GlcNac (3).

Cell lysate would have been tested for active enzyme in the same way, but with the fluorescent substrate 4-MU-N-acetyl-β-D-glucosaminide. Unfortunately, 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]