Difference between revisions of "Part:BBa K2933216"
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<partinfo>BBa_K2933216 parameters</partinfo>
 
<partinfo>BBa_K2933216 parameters</partinfo>
 
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==Usage and Biology===
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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 TMB-2. It encodes a protein which is TMB-2 fused with His tag. The fusion protein is about 27.2 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of TMB-2 .  It is convenient for us to purify our target protein.<br>
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This composite part is made up with seven basic parts, the His tag, T7 promoter, RBS ，Linker h ，Linker f，T7 terminatorand and our target protein TMB-2. It encodes a protein which is TMB-2 fused with His tag. Linker h is from the vector pET-28a, which connects the RBS to His tag sequence.Linker f from vector pGEX-6p-1, contains the thrombin restriction site and T7 tag.The fusion protein is about 27.2 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of TMB-2 .  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 and pET-28b_SUMO 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 pGEX-28a 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:TMB-2-PCR1.png|400px]]<br>
 
   [[File:TMB-2-PCR1.png|400px]]<br>
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'''Figure 1.'''  Left: The PCR result of TMB-2. Right: The verification results by enzyme digestion.<br>
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'''Figure 1.'''  The PCR result of TMB-2.<br>
 
</p>
 
</p>
 
After verification, it was determined that the construction is successful. We converted the plasmid to ''E. coli'' BL21(DE3) for expression and purification.<br>
 
After verification, it was determined that the construction is successful. We converted the plasmid to ''E. coli'' BL21(DE3) for expression and purification.<br>

Latest revision as of 14:04, 23 September 2019


T7 promoter+RBS b+Linker h+His+Linker f+TMB-2+T7 terminator

This part consists of T7 promoter, RBS and protein coding sequence（His+Linker f+TMB-2），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
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 169
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 47
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 47
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This composite part is made up with seven basic parts, the His tag, T7 promoter, RBS ，Linker h ，Linker f，T7 terminatorand and our target protein TMB-2. It encodes a protein which is TMB-2 fused with His tag. Linker h is from the vector pET-28a, which connects the RBS to His tag sequence.Linker f from vector pGEX-6p-1, contains the thrombin restriction site and T7 tag.The fusion protein is about 27.2 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of TMB-2 . It is convenient for us to purify our target protein.

Molecular cloning

First, we used the vector pGEX-28a to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.

TMB-2-PCR1.png
Figure 1. The PCR result of TMB-2.

After verification, it was determined that the construction is successful. We converted the plasmid to E. coli BL21(DE3) for expression and purification.