Difference between revisions of "Part:BBa K2933127"

(→‎Usage and Biology)
(→‎Molecular cloning)
 
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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>
 
[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>
 
===Molecular cloning===
 
===Molecular cloning===
−
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.<br>
+
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>
 
<p style="text-align: center;">
 
<p style="text-align: center;">
 
   [[File:PEDO-1-PCR.png|400px]][[File:PEDO-1-enzyme digestion.png|400px]]<br>
 
   [[File:PEDO-1-PCR.png|400px]][[File:PEDO-1-enzyme digestion.png|400px]]<br>
 
'''Figure 1.'''  Left: The PCR result of PEDO-1. Right: The verification results by enzyme digestion.<br>
 
'''Figure 1.'''  Left: The PCR result of PEDO-1. Right: The verification results by enzyme digestion.<br>
 
</p>
 
</p>

Latest revision as of 11:33, 25 September 2019


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

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


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 256
    Illegal PstI site found at 892
    Illegal PstI site found at 1178
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 256
    Illegal NheI site found at 33
    Illegal PstI site found at 892
    Illegal PstI site found at 1178
  • 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
    Illegal PstI site found at 892
    Illegal PstI site found at 1178
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 256
    Illegal PstI site found at 892
    Illegal PstI site found at 1178
  • 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 PEDO-1. It encodes a protein which is PEDO-1 fused with His-Sumo tag. The fusion protein is about 44.2kD. In order to gain the highly purified target protein, we add His-Sumo tag in N-terminal of PEDO-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.

PEDO-1-PCR.pngPEDO-1-enzyme digestion.png
Figure 1. Left: The PCR result of PEDO-1. Right: The verification results by enzyme digestion.