Difference between revisions of "Part:BBa K2933244"

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===Usage and Biology===
 
===Usage and Biology===
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This composite part is made up with three basic parts, the His-Sumo tag, the cutting site of Prescission Protease and our target protein NDM-23. It encodes a protein which is NDM-23 fused with His-Sumo tag. The fusion protein is about 40.5 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of NDM-23 and combine the two parts with the cutting site of Prescission Protease. The fusion protein can be cut off at the cutting site by Prescission Protease. It is convenient for us to purify our target protein.<br>
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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 NDM-23 and T7 terminator. It encodes a protein which is NDM-23 fused with His-Sumo tag. The fusion protein is about 40.5 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.<br>
  
 
===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.<br>
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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.<br>
 
<p style="text-align: center;">
 
<p style="text-align: center;">
 
   [[File:NDM-23-PCR.png|500px]]<br>
 
   [[File:NDM-23-PCR.png|500px]]<br>
 
'''Figure 1.'''  Left: The PCR result of NDM-23. Right: The verification results by enzyme digestion.<br>
 
'''Figure 1.'''  Left: The PCR result of NDM-23. Right: The verification results by enzyme digestion.<br>
 
</p>
 
</p>

Revision as of 12:48, 22 September 2019


RBS b+Linker h+His+Linker a+Sumo+Linker b+NDM-23+T7 terminator

This part consists of RBS, protein coding sequence(His+Linker a+Sumo+Linker b+NDM-23) 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 1218
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 298
    Illegal BglII site found at 187
    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 NDM-23 and T7 terminator. It encodes a protein which is NDM-23 fused with His-Sumo tag. The fusion protein is about 40.5 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.

NDM-23-PCR.png
Figure 1. Left: The PCR result of NDM-23. Right: The verification results by enzyme digestion.