Difference between revisions of "Part:BBa K2933133"

 
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<partinfo>BBa_K2933133 parameters</partinfo>
 
<partinfo>BBa_K2933133 parameters</partinfo>
 
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===Usage and Biology===
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This composite part is made up with four basic parts, the His tag, the Sumo tag, three cutting sites(NdeI, NheI and BamHI) and our target protein BcII. It encodes a protein which is BcII fused with His-Sumo tag. The fusion protein is about 40.1kD. In order to gain the highly purified target protein, we add His-Sumo tag in N-terminal of BcII 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.<br>
 +
==References==
 +
[1]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===
 +
 +
First,we obtained BcII by PCR.<br>
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<p style="text-align: center;">
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[[File:BcII-194 PCR.jpeg|200px|]]<br>
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'''Figure 1.'''    The PCR result of BcII.<br>
 +
 +
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>
 +
 +
[[File:BcII-194 6p.jpeg|200px|]]<br>
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'''Figure 2.'''  Left:The plasmid of BcII.Right:The verification results by enzyme digestion.<br>
 +
</p>
 +
===Expression and purification===
 +
'''Exploration of expression condition:'''<br>
 +
Take monoclone in the culture plate into LB tube and cultivate in shaking incubator overnight(10-12h) to activate bacteria.
 +
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>
 +
<p style="text-align: center;">
 +
  [[File:BcII.jpeg|200px|]]<br>
 +
</p>

Revision as of 13:49, 24 September 2019


His+Linker a+Sumo+Linker b+BcII-194

This part encodes the fusion protein of His tag, sumo tag and BcII-194 to promote the expression and purification of target protein(BcII-194).


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 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 four basic parts, the His tag, the Sumo tag, three cutting sites(NdeI, NheI and BamHI) and our target protein BcII. It encodes a protein which is BcII fused with His-Sumo tag. The fusion protein is about 40.1kD. In order to gain the highly purified target protein, we add His-Sumo tag in N-terminal of BcII 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.

References

[1]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

Molecular cloning

First,we obtained BcII by PCR.

BcII-194 PCR.jpeg
Figure 1. The PCR result of BcII.
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.
BcII-194 6p.jpeg
Figure 2. Left:The plasmid of BcII.Right:The verification results by enzyme digestion.

Expression and purification

Exploration of expression condition:
Take monoclone in the culture plate into LB tube and cultivate in shaking incubator overnight(10-12h) to activate bacteria. 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.

BcII.jpeg