Difference between revisions of "Part:BBa K2933261"
 
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===Usage and Biology===
 
===Usage and Biology===
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BlaB is a chromosome-encoded MBL produced by Elizabethkingia meningoseptica. It has a broad substrate profile and is produced constitutively. C. meningosepticum is an ubiquitous Gramnegative rod bacterium of clinical relevance because it can cause neonatal meningitis, adult septicemia, and nosocomial infections and is resistant to most β-lactams, including carbapenems.
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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 BlaB-14 and T7 terminator. It encodes a protein which is BlaB-14 fused with His-Sumo tag. The fusion protein is about 40.3 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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This pathogen's multidrug-resistant phenotype and attendant lack of adequate molecular mechanistic data limit the current approaches for its effective management in hospitals and public health settings.<br>
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===Molecular cloning===
 
===Molecular cloning===
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First, 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.
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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.We then extracted plasmids and performed double enzyme digestion verification.<br>
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<p style="text-align: center;">
 
<p style="text-align: center;">
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   [[File:BlaB-14-PCR.png|300px]]<br>
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   [[File:BlaB-14-PCR--.png|400px]]<br>
 
'''Figure 1.'''  (a) The PCR result of BlaB-14. (b) The verification results by enzyme digestion.<br>
 
'''Figure 1.'''  (a) The PCR result of BlaB-14. (b) 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>
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===References===
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[1]González LJ, Vila AJ. Carbapenem resistance in Elizabethkingia meningoseptica is mediated by metallo-β-lactamase BlaB. Antimicrob Agents Chemother. 2012;56(4):1686–1692. doi:10.1128/AAC.05835-11<br>
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[2]Yum, J.H., Lee, E.Y., Hur, SH. et al. J Microbiol. (2010) 48:358.https://doi.org/10.1007/s12275-010-9308-5

Latest revision as of 15:09, 25 September 2019


RBS b+Linker h+His+Linker a+Sumo+Linker b+BlaB-14+T7 terminator

This part consists of RBS, protein coding sequence(His+Linker a+Sumo+Linker b+BlaB-14) 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 1152
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 298
    Illegal BglII site found at 187
    Illegal BglII site found at 958
    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 BlaB-14 and T7 terminator. It encodes a protein which is BlaB-14 fused with His-Sumo tag. The fusion protein is about 40.3 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 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.

BlaB-14-PCR--.png
Figure 1. (a) The PCR result of BlaB-14. (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.

References

[1]González LJ, Vila AJ. Carbapenem resistance in Elizabethkingia meningoseptica is mediated by metallo-β-lactamase BlaB. Antimicrob Agents Chemother. 2012;56(4):1686–1692. doi:10.1128/AAC.05835-11
[2]Yum, J.H., Lee, E.Y., Hur, SH. et al. J Microbiol. (2010) 48:358.https://doi.org/10.1007/s12275-010-9308-5