Difference between revisions of "Part:BBa K1216003"

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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>]]
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<html><table border="1" bordercolor="silver"  style="float:left;margin-top:10px;font-size:12px;font-family:verdana;border-collapse:collapse">
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<tr>
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<th width="220px" >Substrate</th>
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<th width="95px" >Hydrolase</th>
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<th width="120px" >Color, <br>Absorption λ<sub>max</sub></th>
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<th width="145px" >Stock</th>
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<th width="145px" >Liquid culture</th>
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<th width="145px" >Colonies</th>
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<th width="145px" >Response time</th>
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</tr>
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<tr>
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<td bgcolor="#FFFFCC">4-Nitrophenyl- N-acetyl-β-D-glucosaminide (pNP-GluNAc)
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</td>
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<td>NagZ</td>
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<td>Yellow,<br>405 nm</td>
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<td>15 mM in H<sub>2</sub>O</td>
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<td>0.01 mM</td>
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<td>15 mM</td>
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<td>~ 1 minute</td>
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</tr>
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<tr>
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<td bgcolor="#F5CCE0">5-Bromo-6-Chloro-3-indolyl N-acetyl-β-D-glucosaminide (Magenta GluNAc)
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</td>
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<td>NagZ</td>
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<td>Magenta,<br>565 nm</td>
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<td>0.1 M in DMSO</td>
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<td>1 mM, supplemented with BSA</td>
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<td>50 mM, with BSA added</td>
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<td>~ 15 minutes</td>
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</tr>
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<tr>
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<td bgcolor="#F0FAFF">5-Bromo-4-Chloro-3-indolyl N-acetyl-β-D-glucosaminide (X-GluNAc)
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</td>
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<td>NagZ</td>
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<td>Blue,<br>615 nm</td>
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<td>0.1 M in DMSO</td>
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<td>1 mM, supplemented with BSA</td>
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<td>50 mM, with BSA added</td>
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<td>~ 15 minutes</td>
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</tr>
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</table>
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</html>
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<br clear="all">
 
<hr />
 
<hr />
  

Revision as of 16:45, 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.

Colorimetric response

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.

Substrate Hydrolase Color,
Absorption λmax		 Stock Liquid culture Colonies Response time
4-Nitrophenyl- N-acetyl-β-D-glucosaminide (pNP-GluNAc) NagZ Yellow,
405 nm		 15 mM in H2O 0.01 mM 15 mM ~ 1 minute
5-Bromo-6-Chloro-3-indolyl N-acetyl-β-D-glucosaminide (Magenta GluNAc) NagZ Magenta,
565 nm		 0.1 M in DMSO 1 mM, supplemented with BSA 50 mM, with BSA added ~ 15 minutes
5-Bromo-4-Chloro-3-indolyl N-acetyl-β-D-glucosaminide (X-GluNAc) NagZ Blue,
615 nm		 0.1 M in DMSO 1 mM, supplemented with BSA 50 mM, with BSA added ~ 15 minutes

Crosstalk

Figure 26. Liquid cultures of the ΔaesΔgusAΔnagZ Escherichia coli strain overexpressing Aes, GusA, NagZ, PhoA or none in a 96-well plate, with substrates indicated on the left added horizontally.


To ensure orthogonality between the enzyme-substrate reactions used in multi-reporter systems, a crosstalk test was done to make sure that all overexpressed enzymes specifically cleave their assigned substrate.

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]