Difference between revisions of "Part:BBa K2933251"
 
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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 GST tag, the cutting site of Prescission Protease and our target protein CPS-1. It encodes a protein which is CPS-1 fused with GST tag. The fusion protein is about 59.2 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of CPS-1 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 CPS-1 and T7 terminator. It encodes a protein which is CPS-1 fused with His-Sumo tag. The fusion protein is about 45.2 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:CPS-1-PCR.png|500px]]<br>
 
   [[File:CPS-1-PCR.png|500px]]<br>

Latest revision as of 13:12, 22 September 2019


RBS b+Linker h+His+Linker a+Sumo+Linker b+CPS-1+T7 terminator

This part consists of RBS, protein coding sequence(His+Linker a+Sumo+Linker b+CPS-1) 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
    Illegal PstI site found at 934
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 298
    Illegal NheI site found at 75
    Illegal NheI site found at 1278
    Illegal PstI site found at 934
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 298
    Illegal BglII site found at 187
    Illegal BglII site found at 1216
    Illegal BamHI site found at 386
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 298
    Illegal PstI site found at 934
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 298
    Illegal PstI site found at 934
    Illegal AgeI site found at 835
  • 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 CPS-1 and T7 terminator. It encodes a protein which is CPS-1 fused with His-Sumo tag. The fusion protein is about 45.2 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.

CPS-1-PCR.png
Figure 1. Left: The PCR result of CPS-1. Right: 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.