Difference between revisions of "Part:BBa K2933256"
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===Usage and Biology===
 
===Usage and Biology===
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BcII is a type of subclass B metallo-beta-lactamase found in Bacillus anthracis. It can hydrolyzes Beta-lactam antibiotics to make pathogenic bacteria produce drug resistance. The bacterium Bacillus anthracis is responsible for causing anthrax infection, which is often fatal. Because symptoms arising from the bacterial infection are similar to a common cold,misdiagnosis in the early stage is possible and frequent. So the clinical strains carrying it become a great threat to human life and health. Beta-Lactamase inhibitors such as clavulanic acid, sulbactam, and tazobactam are commercially available and can be used with existing antibiotics to cure some antibiotic resistant infections, but these are not effective against MBLs. Although some potential inhibitors have the ability to inhibit it, there are no commercially available inhibitors of it. We use the protein to carry out high-throughput screening to find inhibitors that can inhibit it.<br>
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This composite part is made up with eight basic parts, T7 Ribosome binding sites,the His-Sumo tag, three cutting sites of Prescission Protease, our target protein BcII-194 and T7 terminator. It encodes a protein which is BcII-194 fused with His-Sumo tag. The fusion protein is about 40.1 kD. The fusion protein can be cut off at the cutting sites by Prescission Protease. It is convenient for us to purify our target protein.
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==References==
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Sung-Kun Kim, Mara Demuth, Sara R. Schlesinger, Sung Joon Kim, Jonathan Urbanczyk, Robert W. Shaw & Hyunshun Shin (2016) Inhibition of Bacillusanthracis metallo-β- lactamase by compounds with hydroxamic acid functionality, Journal of Enzyme Inhibition and Medicinal Chemistry, 31:sup4, 132-137, DOI: 10.1080/14756366.2016.1222580<br>
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===Molecular cloning===
 
===Molecular cloning===
  
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'''Figure 1.'''    The PCR result of BcII.<br>
 
'''Figure 1.'''    The PCR result of BcII.<br>
  
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Then 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.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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Then we used the vector PET-28bs to construct our expression plasmid. And then we converted the plasmid constructed to ''E. coli'' DH5α to expand the plasmid largely.After verification, it was determined that the construction is successful. We converted the plasmid to ''E. coli'' BL21(DE3) for expression and purification.<br>
  
 
[[File:BcII-194 6p.jpeg|200px|]]<br>
 
[[File:BcII-194 6p.jpeg|200px|]]<br>
 
'''Figure 2.'''  Left:The plasmid of BcII.Right:The verification results by enzyme digestion.<br>
 
'''Figure 2.'''  Left:The plasmid of BcII.Right:The verification results by enzyme digestion.<br>
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</p>
 
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===Expression and purification===
 
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'''Exploration of expression condition:'''<br>
 
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Take monoclone in the culture plate into LB tube and cultivate in shaking incubator overnight(10-12h) to activate bacteria.
 
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Test the OD600 number of bacteria, then pipe 5-10ul into each new 5 mL LB tube. Don’t forget to add antibiotic into tubes and mark them.Cultivate in shaking incubator for 3-4 hours until the OD600 of bacteria range from 0.6 to 0.8.Set the gradient of condition to explore how to express it best.We use 0.2mM IPTG, 16°C/0.2mM IPTG, 37°C/0.4mM IPTG, 16°C/0.4mM IPTG, 37°C/0.6mM IPTG, 16°C/0.6mM IPTG, 37°C/0.8mM IPTG, 16°C/0.8mM IPTG, 37°Cas different conditions.<br>
 
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<p style="text-align: center;">
 
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  [[File:BcII.jpeg|200px|]]<br>
 
 
</p>
 
</p>

Revision as of 13:36, 22 September 2019


RBS b+Linker h+His+Linker a+Sumo+Linker b+BcII-194+T7 terminator

This part consists of RBS, protein coding sequence(His+Linker a+Sumo+Linker b+BcII-194) and T7 terminator,and the biological module can be build into E.coli for protein expression. This part can be prefaced with promoters of different strengths and types to regulate expression function.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 298
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 298
    Illegal NheI site found at 75
    Illegal NheI site found at 1176
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 298
    Illegal BglII site found at 187
    Illegal BamHI site found at 386
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 298
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 298
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This composite part is made up with eight basic parts, T7 Ribosome binding sites,the His-Sumo tag, three cutting sites of Prescission Protease, our target protein BcII-194 and T7 terminator. It encodes a protein which is BcII-194 fused with His-Sumo tag. The fusion protein is about 40.1 kD. The fusion protein can be cut off at the cutting sites by Prescission Protease. It is convenient for us to purify our target protein.

Molecular cloning

First,we obtained BcII by PCR.

BcII-194 PCR.jpeg
Figure 1. The PCR result of BcII.
Then we used the vector PET-28bs to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.
BcII-194 6p.jpeg
Figure 2. Left:The plasmid of BcII.Right:The verification results by enzyme digestion.