Difference between revisions of "Part:BBa K2933207"

 
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<partinfo>BBa_K2933207 parameters</partinfo>
 
<partinfo>BBa_K2933207 parameters</partinfo>
 
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==Usage and Biology===
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This composite part is made up with three basic parts, the His tag, T7 promoter, RBS and our target protein Myxo. It encodes a protein which is Myxo fused with His tag. The fusion protein is about 27.9 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of Myxo .  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 13:44, 21 September 2019


T7 promoter+RBS b+Linker h+His+Linker f+MYX-1+T7 terminator

This part consists of T7 promoter, RBS and protein coding sequence（His+Linker f+Myxo），and the biological module can be built into E.coli for protein expression.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 47
    Illegal PstI site found at 284
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 169
    Illegal PstI site found at 284
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 47
    Illegal PstI site found at 284
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 47
    Illegal PstI site found at 284
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology=

This composite part is made up with three basic parts, the His tag, T7 promoter, RBS and our target protein Myxo. It encodes a protein which is Myxo fused with His tag. The fusion protein is about 27.9 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of Myxo . 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.