Difference between revisions of "Part:BBa K1216003"

Revision as of 22:07, 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 λ_max	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

To ensure specificity of the enzyme-substrate pairs used in Colisweeper, a crosstalk test was done to make sure that all overexpressed enzymes specifically cleave their assigned substrate.

Figure 26.2. 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 26.1.). As Figure 26.2. shows, the overexpressed hydrolases cleave only the substrates they were expected to.

Figure 26.1. Expected outcome. Added substrates should be specifically cleaved by their hydrolases.

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.
[http://www.inalcopharm.com/cart.php?m=product_detail&p=6 Inalcopharm datasheet]

@@ Line 78: / Line 78: @@
 ===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 Colisweeper, a crosstalk test was done to make sure that all overexpressed enzymes specifically cleave their assigned substrate.</p>
+[[File:Crosstalk_ethz.png|thumb|right|450px| <b>Figure 26.2.</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 26.1.). As Figure 26.2. shows, the overexpressed hydrolases cleave only the substrates they were expected to.
+</p>
+[[File:CrosstalkExpected.png|frame|center| <b>Figure 26.1. Expected outcome.</b> Added substrates should be specifically cleaved by their hydrolases.]]
 <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>
 ===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.
 # [http://www.inalcopharm.com/cart.php?m=product_detail&p=6 Inalcopharm datasheet]