Difference between revisions of "Part:BBa K2933248"

 
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<partinfo>BBa_K2933248 parameters</partinfo>
 
<partinfo>BBa_K2933248 parameters</partinfo>
 
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===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 Myxo. It encodes a protein which is Myxo fused with GST tag. The fusion protein is about 53.9 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of Myxo 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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===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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<p style="text-align: center;">
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  [[File:T--TJUSLS China--MYX-1 PCRmeiqie.png|300px]]  [[File:T--TJUSLS China--MYX-1 meiqie.png|300px]]<br>
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'''Figure 1.'''  Left: The PCR result of MYX-1. Right: 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>

Revision as of 11:10, 21 September 2019


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

This part consists of RBS, protein coding sequence(His+Linker a+Sumo+Linker b+Myxo) 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 477
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 298
    Illegal NheI site found at 75
    Illegal NheI site found at 1191
    Illegal PstI site found at 477
  • 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
    Illegal PstI site found at 477
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 298
    Illegal PstI site found at 477
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This composite part is made up with three basic parts, the GST tag, the cutting site of Prescission Protease and our target protein Myxo. It encodes a protein which is Myxo fused with GST tag. The fusion protein is about 53.9 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of Myxo 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.

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.

T--TJUSLS China--MYX-1 PCRmeiqie.png T--TJUSLS China--MYX-1 meiqie.png
Figure 1. Left: The PCR result of MYX-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.