Difference between revisions of "Part:BBa K2933017"

 
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<partinfo>BBa_K2933017 parameters</partinfo>
 
<partinfo>BBa_K2933017 parameters</partinfo>
 
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===Usage and Biology===
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PST-1 is a type of subclass B1 metal beta-lactamases. The beta lactamases of subclass B1 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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===References===
 +
 +
 +
===Molecular cloning===
 +
First, we used the vector pET28B-Sumo to construct our expression plasmid. And then we converted the plasmid constructed to ''E. coli'' DH5α to expand the plasmid largely.<br>
 +
<p style="text-align: center;">
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  [[File:PST-1-PCR.png]]<br>
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'''Figure 1.'''  a: The PCR result of PST-1. b: The verification results by enzyme digestion.<br>
 +
</p>
 +
After verification, it was determined that the construction is successful. We converted the plasmid to ''E. coli'' BL21(DE3) for expression and purification.<br>
 +
 +
===Expression and purification===
 +
'''Pre-expression:'''<br>
 +
The bacteria were cultured in 5mL LB liquid medium with ampicillin(100 μg/mL final concentration) in 37℃ overnight.<br>
 +
'''Massive expressing:'''<br>
 +
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>
 +
 +
'''Affinity Chromatography:'''<br>
 +
We used the Ni-sepharose to purify the target protein. The Ni-sepharose can combine specifically with the His tag fused with target protein. <br>
 +
* First, wash the column with water for 10 minutes. Change to Ni-binding buffer for another 10 minutes and balance the Ni column.<br>
 +
* Second, add the protein solution to the column, let it flow naturally and bind to the column. <br>
 +
* Third, add Ni-Washing buffer several times and let it flow. Take 5ul of wash solution and test with Coomassie Brilliant Blue. Stop washing when it doesn’t turn blue.<br>
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* Forth, add Ni-Elution buffer several times. Check as above.<br>
 +
* Fifth, collect the eluted proteins for further operation. <br>
 +
<p style="text-align: center;">
 +
    [[File:T--TJUSLS China--PST-1 Ni.png]]<br>
 +
 +
'''Figure 2.'''  The result of SDS-page.<br>
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</p>

Latest revision as of 08:41, 24 September 2019


subclass B1 metallo-beta-lactamase PST-1, codon optimized in E. coli

This part encodes a protein called PST-1, which is a metallo-beta-lactamase of subclass B1.

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]


Usage and Biology

PST-1 is a type of subclass B1 metal beta-lactamases. The beta lactamases of subclass B1 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.

References

Molecular cloning

First, we used the vector pET28B-Sumo to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.

PST-1-PCR.png
Figure 1. a: The PCR result of PST-1. b: 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 Ni-sepharose to purify the target protein. The Ni-sepharose can combine specifically with the His tag fused with target protein.

  • First, wash the column with water for 10 minutes. Change to Ni-binding buffer for another 10 minutes and balance the Ni column.
  • Second, add the protein solution to the column, let it flow naturally and bind to the column.
  • Third, add Ni-Washing buffer several times and let it flow. Take 5ul of wash solution and test with Coomassie Brilliant Blue. Stop washing when it doesn’t turn blue.
  • Forth, add Ni-Elution buffer several times. Check as above.
  • Fifth, collect the eluted proteins for further operation.

T--TJUSLS China--PST-1 Ni.png
Figure 2. The result of SDS-page.