Difference between revisions of "Part:BBa K2933128"

 
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<partinfo>BBa_K2933128 parameters</partinfo>
 
<partinfo>BBa_K2933128 parameters</partinfo>
 
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===Usage and Biology===
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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 CPS-1. It encodes a protein which is CPS-1 fused with His-Sumo tag. The fusion protein is about 45.2kD. In order to gain the highly purified target protein, we add His-Sumo tag in N-terminal of CPS-1 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.
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===References===
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[1]Dereje Dadi Gudeta, Valeria Bortolaia,The Soil Microbiota Harbors a Diversity of Carbapenem-Hydrolyzing β-Lactamases of Potential Clinical Relevance[J]，Antimicrobial Agents and chemotherapy,January 2016<br>
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===Molecular cloning===
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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.<br>
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<p style="text-align: center;">
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[[File:CPS-1-PCR.png|400px]][[File:CPS-1-enzyme digestion.png|400px]]<br>
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'''Figure 1.'''  a: The PCR result of CPS-1. b: The verification results by enzyme digestion.<br>
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</p>
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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 11:34, 25 September 2019


His+Linker a+Sumo+Linker b+CPS-1

This part encodes the fusion protein of His tag, sumo tag and CPS-1 to promote the expression and purification of target protein(CPS-1).


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 256
    Illegal PstI site found at 892
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 256
    Illegal NheI site found at 33
    Illegal PstI site found at 892
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 256
    Illegal BglII site found at 145
    Illegal BglII site found at 1174
    Illegal BamHI site found at 344
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 256
    Illegal PstI site found at 892
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 256
    Illegal PstI site found at 892
    Illegal AgeI site found at 793
  • 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 CPS-1. It encodes a protein which is CPS-1 fused with His-Sumo tag. The fusion protein is about 45.2kD. In order to gain the highly purified target protein, we add His-Sumo tag in N-terminal of CPS-1 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]Dereje Dadi Gudeta, Valeria Bortolaia,The Soil Microbiota Harbors a Diversity of Carbapenem-Hydrolyzing β-Lactamases of Potential Clinical Relevance[J]，Antimicrobial Agents and chemotherapy,January 2016

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.

CPS-1-PCR.pngCPS-1-enzyme digestion.png
Figure 1. a: The PCR result of CPS-1. 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.