Difference between revisions of "Part:BBa K1216003"

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==Characterization==
 
==Characterization==
  
−
<b>The final construct was sequenced.</b>
+
<b>The final construct was sequenced.</b><br>
 +
 
 +
Jump to: <br>
 +
*[[Part:BBa_K1216003#Colorimetric response|Colorimetric response]]
 +
*[[Part:BBa_K1216003#Crosstalk|Crosstalk]]
 +
<br>
 +
 
 
===Colorimetric response===
 
===Colorimetric response===
−
[[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.]]
+
[[File:NagZ3substrates.JPG|thumb|right|300px|<b>Figure 1.  Liquid cultures from the triple knockout <i>E.Coli</i> strain 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.
−
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).  
+
To test the functionality of the enzyme, a liquid culture of the Δ''aes''Δ''gusA''Δ''nagZ'' ''Escherichia coli'' strain overexpressing NagZ was incubated with its chromogenic substrates magenta-GlcNac (produces magenta color), pNP-GlcNac (produces yellow color) and X-GlcNac (produces blue color).  
 
[[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>]]
 
[[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 (Figure 4). 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>]]
 
<html><table border="1" bordercolor="silver"  style="float:left;margin-top:10px;font-size:12px;font-family:verdana;border-collapse:collapse">
 
<html><table border="1" bordercolor="silver"  style="float:left;margin-top:10px;font-size:12px;font-family:verdana;border-collapse:collapse">
 
<tr>
 
<tr>
−
<th width="220px" >Substrate</th>
 
 
<th width="95px" >Hydrolase</th>
 
<th width="95px" >Hydrolase</th>
−
<th width="120px" >Color, <br>Absorption λ<sub>max</sub></th>
+
<th width="220px" >Substrate</th>
−
<th width="145px" >Stock</th>
+
<th width="120px" >Absorption λ<sub>max</sub> or Excitation/Emission</th>
−
<th width="145px" >Liquid culture</th>
+
<th width="145px" >Stock solution</th>
−
<th width="145px" >Colonies</th>
+
<th width="145px" >Liquid culture: end concentration</th>
 +
<th width="145px" >Colonies: 1.5 &mu;l of substrate solution</th>
 
<th width="145px" >Response time</th>
 
<th width="145px" >Response time</th>
 
</tr>
 
</tr>
 
<tr>
 
<tr>
 +
<td rowspan="3" align="center">NagZ</td>
 
<td bgcolor="#FFFFCC">4-Nitrophenyl- N-acetyl-β-D-glucosaminide (pNP-GluNAc)
 
<td bgcolor="#FFFFCC">4-Nitrophenyl- N-acetyl-β-D-glucosaminide (pNP-GluNAc)
 
</td>
 
</td>
−
<td>NagZ</td>
 
 
<td>Yellow,<br>405 nm</td>
 
<td>Yellow,<br>405 nm</td>
 
<td>15 mM in H<sub>2</sub>O</td>
 
<td>15 mM in H<sub>2</sub>O</td>
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<td bgcolor="#F5CCE0">5-Bromo-6-Chloro-3-indolyl N-acetyl-β-D-glucosaminide (Magenta GluNAc)
 
<td bgcolor="#F5CCE0">5-Bromo-6-Chloro-3-indolyl N-acetyl-β-D-glucosaminide (Magenta GluNAc)
 
</td>
 
</td>
−
<td>NagZ</td>
 
 
<td>Magenta,<br>565 nm</td>
 
<td>Magenta,<br>565 nm</td>
 
<td>0.1 M in DMSO</td>
 
<td>0.1 M in DMSO</td>
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<td bgcolor="#F0FAFF">5-Bromo-4-Chloro-3-indolyl N-acetyl-β-D-glucosaminide (X-GluNAc)
 
<td bgcolor="#F0FAFF">5-Bromo-4-Chloro-3-indolyl N-acetyl-β-D-glucosaminide (X-GluNAc)
 
</td>
 
</td>
−
<td>NagZ</td>
 
 
<td>Blue,<br>615 nm</td>
 
<td>Blue,<br>615 nm</td>
 
<td>0.1 M in DMSO</td>
 
<td>0.1 M in DMSO</td>
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</table>
 
</table>
 
</html>
 
</html>
−
<br clear="all">
+
<br clear="all"><br>
 
<hr />
 
<hr />
  
 
===Crosstalk===
 
===Crosstalk===
−
[[File:Crosstalk_ethz.png|thumb|left|450px| <b>Figure 26.</b> Liquid cultures of the Δ''aes''Δ''gusA''Δ''nagZ'' <i>Escherichia coli</i> strain overexpressing Aes, GusA, NagZ, PhoA or none in a 96-well plate, with substrates indicated on the left added horizontally.]]
+
<p align="justify">To ensure specificity of the enzyme-substrate pairs used in [http://2013.igem.org/Team:ETH_Zurich Colisweeper] (ETH Zurich 2013), a crosstalk test was done to make sure that all overexpressed enzymes specifically cleave their assigned substrate.</p>
 +
 
 +
[[File:Crosstalk.png|thumb|right|450px| <b>Figure 6.</b> Liquid cultures of the Δ''aes''Δ''gusA''Δ''nagZ'' <i>Escherichia coli</i> strain overexpressing Aes, GusA, NagZ or none in a 96-well plate, with substrates indicated on the left added horizontally.]]
 +
 
 +
<p align="justify">This crosstalk test was done in a 96-well plate, each well containing 200 μl from liquid cultures of our Δ''aes''Δ''gusA''Δ''nagZ'' <i>Escherichia coli</i> strain overexpressing either Aes, GusA, NagZ or none, each distributed among the column-wells of the plate. Horizontally, the chromogenic substrates were pipetted to the liquid cultures in the same order as their corresponding hydrolase. If specificity of the chosen enzyme-substrates pairs were given, we would expect an output as shown in the figure below (Figure 5). As Figure 6 shows, the overexpressed hydrolases cleave only the substrates they were expected to.
 +
</p>
 +
[[File:CrosstalkExpected.png|frame|center| <b>Figure 5. Expected outcome.</b> Added substrates should be specifically cleaved by their hydrolases.]]
 
<br clear="all"/>
 
<br clear="all"/>
−
<p align="justify">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.</p>
+
<hr />
  
 
===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]

Latest revision as of 16:36, 30 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.

Jump to:


Colorimetric response

Figure 1. Liquid cultures from the triple knockout E.Coli strain 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 the ΔaesΔgusAΔnagZ Escherichia coli strain 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 (Figure 4). Unfortunately, this did not work until the wiki freeze.

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

Hydrolase Substrate Absorption λmax or Excitation/Emission Stock solution Liquid culture: end concentration Colonies: 1.5 μl of substrate solution Response time
NagZ 4-Nitrophenyl- N-acetyl-β-D-glucosaminide (pNP-GluNAc) 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) 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) Blue,
615 nm		 0.1 M in DMSO 1 mM, supplemented with BSA 50 mM, with BSA added ~ 15 minutes


Crosstalk

To ensure specificity of the enzyme-substrate pairs used in [http://2013.igem.org/Team:ETH_Zurich Colisweeper] (ETH Zurich 2013), a crosstalk test was done to make sure that all overexpressed enzymes specifically cleave their assigned substrate.

Figure 6. Liquid cultures of the ΔaesΔgusAΔnagZ Escherichia coli strain overexpressing Aes, GusA, NagZ or none in a 96-well plate, with substrates indicated on the left added horizontally.

This crosstalk test was done in a 96-well plate, each well containing 200 μl from liquid cultures of our ΔaesΔgusAΔnagZ Escherichia coli strain overexpressing either Aes, GusA, NagZ or none, each distributed among the column-wells of the plate. Horizontally, the chromogenic substrates were pipetted to the liquid cultures in the same order as their corresponding hydrolase. If specificity of the chosen enzyme-substrates pairs were given, we would expect an output as shown in the figure below (Figure 5). As Figure 6 shows, the overexpressed hydrolases cleave only the substrates they were expected to.

Figure 5. Expected outcome. Added substrates should be specifically cleaved by their hydrolases.


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]