Difference between revisions of "Part:BBa K2933013"

 
 
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<partinfo>BBa_K2933013 short</partinfo>
 
<partinfo>BBa_K2933013 short</partinfo>
  
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This part encodes a protein called VIM-66, which is a metallo-beta-lactamase of subclass B1.
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This part encodes a protein called SHD, which is a metallo-beta-lactamase of subclass B1.
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<!-- Add more about the biology of this part here
 
 
===Usage and Biology===
 
===Usage and Biology===
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SHD is a type of subclass B metal beta-lactamases. The beta lactamases of the SHD family can hydrolyze almost all available beta lactam antibiotics (except aztreonam) clinically, including the broad-spectrum antibiotic carbapenems. Because of the extensive substrate profile of this enzyme, the clinical strains carrying it become a great threat to human life and health.<br>
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K2933013 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K2933013 SequenceAndFeatures</partinfo>
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===Molecular cloning===
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First, we used the vector pGEX-6p-1 to construct our expression plasmid. And then we converted the plasmid constructed to ''E. coli'' DH5α to expand the plasmid largely.<br>
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<p style="text-align: center;">
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  [[File:SHD-PCR.png|500px]]<br>
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'''Figure 1.'''  Left: The PCR result of SHD. Right: The verification results by enzyme digestion.<br>
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</p>
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After verification, it was determined that the construction is successful. We converted the plasmid to ''E. coli'' BL21(DE3) for expression and purification.<br>
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===Expression and purification===
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'''Pre-expression:'''<br>
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The bacteria were cultured in 5mL LB liquid medium with ampicillin(100 μg/mL final concentration) in 37℃ overnight.<br>
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'''Massive expressing:'''<br>
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After taking samples, we transfered them into 1L LB medium and add antibiotic to 100 μg/mL final concentration. Grow them up in 37°C shaking incubator. Grow until an OD 600 nm of 0.8 to 1.2 (roughly 3-4 hours). Induce the culture to express protein by adding 1 mM IPTG (isopropylthiogalactoside, MW 238 g/mol). Put the liter flasks in 16°C shaking incubator for 16h.<br>
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'''Affinity Chromatography:'''<br>
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We used the GST Agarose to purify the target protein. The GST Agarose can combine specifically with the GST tag fused with target protein. <br>
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* First, wash the column with GST-binding buffer for 10 minutes to balance the GST column.<br>
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* Second, add the protein solution to the column, let it flow naturally and bind to the column.<br>
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* Third, add GST-Washing buffer several times and let it flow. Take 10μl of wash solution and test with Coomassie Brilliant Blue. Stop washing when it doesn’t turn blue.<br>
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* Forth, add 400μL Prescission Protease (1mg/mL) to the agarose. Digest for 16 hours in 4℃.
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* Fifth, add GST-Elution buffer several times. Check as above. Collect the eluted proteins for further operation.<br>
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<p style="text-align: center;">
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    [[File:T--TJUSLS China--SHD GST.jpg|400px]]<br>
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'''Figure 2.'''  The result of SDS-page.<br>
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</p>
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==References==
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Latest revision as of 10:26, 21 September 2019


subclass B1 metallo-beta-lactamase SHD, codon optimized in E. coli

This part encodes a protein called SHD, which is a metallo-beta-lactamase of subclass B1.


Usage and Biology

SHD is a type of subclass B metal beta-lactamases. The beta lactamases of the SHD family can hydrolyze almost all available beta lactam antibiotics (except aztreonam) clinically, including the broad-spectrum antibiotic carbapenems. Because of the extensive substrate profile of this enzyme, the clinical strains carrying it become a great threat to human life and health.

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]

Molecular cloning

First, we used the vector pGEX-6p-1 to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.

SHD-PCR.png
Figure 1. Left: The PCR result of SHD. Right: The verification results by enzyme digestion.

After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.


Expression and purification

Pre-expression:
The bacteria were cultured in 5mL LB liquid medium with ampicillin(100 μg/mL final concentration) in 37℃ overnight.
Massive expressing:
After taking samples, we transfered them into 1L LB medium and add antibiotic to 100 μg/mL final concentration. Grow them up in 37°C shaking incubator. Grow until an OD 600 nm of 0.8 to 1.2 (roughly 3-4 hours). Induce the culture to express protein by adding 1 mM IPTG (isopropylthiogalactoside, MW 238 g/mol). Put the liter flasks in 16°C shaking incubator for 16h.

Affinity Chromatography:
We used the GST Agarose to purify the target protein. The GST Agarose can combine specifically with the GST tag fused with target protein.

  • First, wash the column with GST-binding buffer for 10 minutes to balance the GST column.
  • Second, add the protein solution to the column, let it flow naturally and bind to the column.
  • Third, add GST-Washing buffer several times and let it flow. Take 10μl of wash solution and test with Coomassie Brilliant Blue. Stop washing when it doesn’t turn blue.
  • Forth, add 400μL Prescission Protease (1mg/mL) to the agarose. Digest for 16 hours in 4℃.
  • Fifth, add GST-Elution buffer several times. Check as above. Collect the eluted proteins for further operation.

T--TJUSLS China--SHD GST.jpg
Figure 2. The result of SDS-page.

References