Difference between revisions of "Part:BBa K2933138"

(→‎Usage and Biology)
(→‎Molecular cloning)
 
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===Molecular cloning===
 
===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.We then extracted plasmids and performed double enzyme digestion verification.<br>
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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.We then extracted plasmids and performed double enzyme digestion verification.<br>
 
<p style="text-align: center;">
 
<p style="text-align: center;">
 
   [[File:BlaB-14-PCR.png|300px]]<br>
 
   [[File:BlaB-14-PCR.png|300px]]<br>
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</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>
 +
 
===References===
 
===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<br>
 
[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>
 
[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
 
[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 11:46, 25 September 2019


His+Linker a+Sumo+Linker b+BlaB-14

This part encodes the fusion protein of His tag, sumo tag and BlaB-14 to promote the expression and purification of target protein(BlaB-14).


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 256
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 256
    Illegal NheI site found at 33
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 256
    Illegal BglII site found at 145
    Illegal BglII site found at 916
    Illegal BamHI site found at 344
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 256
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 256
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This composite part is made up with five basic parts, the His tag, the Sumo tag, three cutting sites(NdeI, NheI and BamHI) and our target protein BlaB-14. It encodes a protein which is BlaB-14 fused with His-Sumo tag. The fusion protein is about 40.3kD. In order to gain the highly purified target protein, we add His-Sumo tag in N-terminal of BlaB-14 and combine the three parts with these three cutting sites. The fusion protein can be cut off at the cutting site BamHI. It is convenient for us to purify our target protein.

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.We then extracted plasmids and performed double enzyme digestion verification.

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