Difference between revisions of "Part:BBa K2933020"
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This part encodes a protein called IMP-71, which is a metallo-beta-lactamase of subclass B1.
 
This part encodes a protein called IMP-71, which is a metallo-beta-lactamase of subclass B1.
  
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Bacteria with IMP-type enzymes have spread through out the world, and the IMP group now has more than 50 variants
 
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These enzymes possess a broad substrate specificity and a high affinity for cephalosporins and carbapenems but a low activity toward temocillin
 
  
  
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===Usage and Biology===
 
===Usage and Biology===
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IMP-71 is a type of subclass B metal beta-lactamases, which is derived from IMP-1 mutation. The beta lactamases of the IMP 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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Bacteria with IMP-type enzymes have spread through out the world, and the IMP group now has more than 50 variants
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==References==
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These enzymes possess a broad substrate specificity and a high affinity for cephalosporins and carbapenems but a low activity toward temocillin<br>
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===References===
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[1]Purification, crystallization and preliminary X-ray analysis of IMP-18, a class B carbapenemase from Pseudomonas aeruginosa.Furuyama T, Ishii Y, Ohya N, Tateda K, Hanson ND, Shimizu-Ibuka A.Acta Crystallogr Sect F Struct Biol Cryst Commun. 2013 Dec;69(Pt 12):1397-400.
  
 
===Molecular cloning===
 
===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.<br>
 
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>
 
<p style="text-align: center;">
 
<p style="text-align: center;">
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   [[File:IMP-71-PCR.png|200px]]<br>
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   [[File:IMP-71-PCR.png|400px]]<br>
 
'''Figure 1.'''  Left: The PCR result of IMP-71. Right: The verification results by enzyme digestion.<br>
 
'''Figure 1.'''  Left: The PCR result of IMP-71. Right: The verification results by enzyme digestion.<br>
 
</p>
 
</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>
 
After verification, it was determined that the construction is successful. We converted the plasmid to ''E. coli'' BL21(DE3) for expression and purification.<br>

Latest revision as of 14:56, 23 September 2019


subclass B1 metallo-beta-lactamase IMP-71, codon optimized in E. coli

This part encodes a protein called IMP-71, which is a metallo-beta-lactamase of subclass B1.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 724
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 724
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 724
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 724
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

Bacteria with IMP-type enzymes have spread through out the world, and the IMP group now has more than 50 variants These enzymes possess a broad substrate specificity and a high affinity for cephalosporins and carbapenems but a low activity toward temocillin

References

[1]Purification, crystallization and preliminary X-ray analysis of IMP-18, a class B carbapenemase from Pseudomonas aeruginosa.Furuyama T, Ishii Y, Ohya N, Tateda K, Hanson ND, Shimizu-Ibuka A.Acta Crystallogr Sect F Struct Biol Cryst Commun. 2013 Dec;69(Pt 12):1397-400.

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.

IMP-71-PCR.png
Figure 1. Left: The PCR result of IMP-71. 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.