Difference between revisions of "Part:BBa K3731001"
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The pQE30-mazE plasmid containing a 21 residue N-terminal tag, six of which are histidines, was transformed into E. coli MC4100ΔmazEF-lacI (relA) and cells were grown in 1 l of M9 minimal medium at 310 K supplemented with 15N-labeled NH4Cl and/or 13C-labeled glucose, respectively, and 100 μg/ml ampicillin. At an OD600 of 0.8 protein expression was induced with 1 mM IPTG and after additional 5 h of incubation the cells were harvested by centrifugation (30 min/4.000 g/277 K) and resuspended in 50 mM K2HPO4, 300 mM NaCl, 10 mM imidazole, pH 8.0. After sonication the suspension was again centrifuged (60 min/15.000 g/277 K) and the supernatant filtered through a 0.45 μm sterile filter prior to loading it onto a Ni-CAM column (Sigma Aldrich, St. Louis, MO, USA), pre-equilibrated in 50 mM K2HPO4, 300 mM NaCl, 10 mM imidazole, pH 8.0. The protein was eluted from the column with 50 mM K2HPO4, 300 mM NaCl, 500 mM imidazole, pH 8.0 with a 10–500 mM imidazole gradient as a single peak at an approximate concentration of 100-mM imidazole. The fractions containing the protein were combined and dialyzed first against 2 l of distilled water followed by 1 l of 20 mM KH2PO4, 100 mM NaCl, pH 6.5 as nuclear magnetic resonance (NMR) buffer. In a final step the concentrated protein was heated up to 358–363 K for 2 min and slowly cooled down to room temperature. This heat treatment makes EcMazE more stable for long-term storage at room temperature, since it denatures contaminating proteins, such as proteases, while EcMazE can be refolded upon heat denaturation like other bacterial antitoxins.
 
The pQE30-mazE plasmid containing a 21 residue N-terminal tag, six of which are histidines, was transformed into E. coli MC4100ΔmazEF-lacI (relA) and cells were grown in 1 l of M9 minimal medium at 310 K supplemented with 15N-labeled NH4Cl and/or 13C-labeled glucose, respectively, and 100 μg/ml ampicillin. At an OD600 of 0.8 protein expression was induced with 1 mM IPTG and after additional 5 h of incubation the cells were harvested by centrifugation (30 min/4.000 g/277 K) and resuspended in 50 mM K2HPO4, 300 mM NaCl, 10 mM imidazole, pH 8.0. After sonication the suspension was again centrifuged (60 min/15.000 g/277 K) and the supernatant filtered through a 0.45 μm sterile filter prior to loading it onto a Ni-CAM column (Sigma Aldrich, St. Louis, MO, USA), pre-equilibrated in 50 mM K2HPO4, 300 mM NaCl, 10 mM imidazole, pH 8.0. The protein was eluted from the column with 50 mM K2HPO4, 300 mM NaCl, 500 mM imidazole, pH 8.0 with a 10–500 mM imidazole gradient as a single peak at an approximate concentration of 100-mM imidazole. The fractions containing the protein were combined and dialyzed first against 2 l of distilled water followed by 1 l of 20 mM KH2PO4, 100 mM NaCl, pH 6.5 as nuclear magnetic resonance (NMR) buffer. In a final step the concentrated protein was heated up to 358–363 K for 2 min and slowly cooled down to room temperature. This heat treatment makes EcMazE more stable for long-term storage at room temperature, since it denatures contaminating proteins, such as proteases, while EcMazE can be refolded upon heat denaturation like other bacterial antitoxins.
 
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<p>Besides, the structure of mazE antitoxin is drawn by PyMol as shown below.
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<p>Besides, the structure of mazE antitoxin is drawn by PyMol as is shown below.
  
 
[[File:T--Nanjing-China--mazE.png|800px|thumb|center|Figure1 Structure of mazE]]  
 
[[File:T--Nanjing-China--mazE.png|800px|thumb|center|Figure1 Structure of mazE]]  
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<p>Reference:
 
<p>Reference:
−
Zorzini, Buts et al. Escherichia coli antitoxin MazE as transcription factor: insights into MazE-DNA binding, Nucleic Acids Research, Volume 43, Issue 2, 30 January 2015, Pages 1241–1256</p>
+
</p>
 +
<p>Zorzini, Buts et al. Escherichia coli antitoxin MazE as transcription factor: insights into MazE-DNA binding, Nucleic Acids Research, Volume 43, Issue 2, 30 January 2015, Pages 1241–1256</p>
 +
</p>

Revision as of 13:08, 14 September 2021


mazE

The mazE codes a kind of antitoxin protein, and it is connected with ppk1 and vgb, which can help the bacteria grow longer and produce more polyP. We use PAGE assay to verify the completion of the experiment.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


iGEM2021_Nanjing-China Experiment

Group: Nanjing-China 2021
Author: Hao Yin

Toxin-antitoxin (TA) modules are pairs of genes essential for bacterial regulation upon environmental stresses, among which mazE is an important antitoxin.

Expression and purification of full-length EcMazE are shown here: The pQE30-mazE plasmid containing a 21 residue N-terminal tag, six of which are histidines, was transformed into E. coli MC4100ΔmazEF-lacI (relA) and cells were grown in 1 l of M9 minimal medium at 310 K supplemented with 15N-labeled NH4Cl and/or 13C-labeled glucose, respectively, and 100 μg/ml ampicillin. At an OD600 of 0.8 protein expression was induced with 1 mM IPTG and after additional 5 h of incubation the cells were harvested by centrifugation (30 min/4.000 g/277 K) and resuspended in 50 mM K2HPO4, 300 mM NaCl, 10 mM imidazole, pH 8.0. After sonication the suspension was again centrifuged (60 min/15.000 g/277 K) and the supernatant filtered through a 0.45 μm sterile filter prior to loading it onto a Ni-CAM column (Sigma Aldrich, St. Louis, MO, USA), pre-equilibrated in 50 mM K2HPO4, 300 mM NaCl, 10 mM imidazole, pH 8.0. The protein was eluted from the column with 50 mM K2HPO4, 300 mM NaCl, 500 mM imidazole, pH 8.0 with a 10–500 mM imidazole gradient as a single peak at an approximate concentration of 100-mM imidazole. The fractions containing the protein were combined and dialyzed first against 2 l of distilled water followed by 1 l of 20 mM KH2PO4, 100 mM NaCl, pH 6.5 as nuclear magnetic resonance (NMR) buffer. In a final step the concentrated protein was heated up to 358–363 K for 2 min and slowly cooled down to room temperature. This heat treatment makes EcMazE more stable for long-term storage at room temperature, since it denatures contaminating proteins, such as proteases, while EcMazE can be refolded upon heat denaturation like other bacterial antitoxins.

Besides, the structure of mazE antitoxin is drawn by PyMol as is shown below.

Figure1 Structure of mazE

The mazE protein is such a useful antitoxin that it can help bacteria grow longer and survive harsher environment, thus producing more polyP. Thus, we insert mazE gene into E. coli genome to increase the productivity.


Reference:

Zorzini, Buts et al. Escherichia coli antitoxin MazE as transcription factor: insights into MazE-DNA binding, Nucleic Acids Research, Volume 43, Issue 2, 30 January 2015, Pages 1241–1256

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