Difference between revisions of "Part:BBa K2933204"
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<partinfo>BBa_K2933204 parameters</partinfo>
 
<partinfo>BBa_K2933204 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 SPG-1. It encodes a protein which is SPG-1 fused with His tag. The fusion protein is about 30.2 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of SPG-1.  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 terminator and our target protein SPG-1. It encodes a protein which is SPG-1 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 30.2 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of SPG-1.  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 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:SPG-PCR1.png]]<br>
 
   [[File:SPG-PCR1.png]]<br>
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'''Figure 1.'''   a: The PCR result of SPG. <br>
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'''Figure 1.''' The PCR result of SPG. <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>
 +
 
===Expression and purification===
 
===Expression and purification===
 
'''Pre-expression:'''<br>
 
'''Pre-expression:'''<br>
 
The bacteria were cultured in 5mL LB liquid medium with ampicillin(100 μg/mL final concentration) in 37℃ overnight.<br>
 
The bacteria were cultured in 5mL LB liquid medium with ampicillin(100 μg/mL final concentration) in 37℃ overnight.<br>
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===References===
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[1]The Soil Microbiota Harbors a Diversity of Carbapenem-Hydrolyzing β-Lactamases of Potential Clinical Relevance. Gudeta DD, Bortolaia V, Amos G, Wellington EM, Brandt KK, Poirel L, Nielsen JB, Westh H, Guardabassi L. Antimicrob Agents Chemother. 2015 Oct 19;60(1):151-60.

Latest revision as of 14:00, 23 September 2019


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

This part consists of T7 promoter, RBS and protein coding sequence（His+Linker f+SPG-1），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 terminator and our target protein SPG-1. It encodes a protein which is SPG-1 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 30.2 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of SPG-1. 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.

SPG-PCR1.png
Figure 1. The PCR result of SPG.

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

Expression and purification

Pre-expression:
The bacteria were cultured in 5mL LB liquid medium with ampicillin(100 μg/mL final concentration) in 37℃ overnight.

References

[1]The Soil Microbiota Harbors a Diversity of Carbapenem-Hydrolyzing β-Lactamases of Potential Clinical Relevance. Gudeta DD, Bortolaia V, Amos G, Wellington EM, Brandt KK, Poirel L, Nielsen JB, Westh H, Guardabassi L. Antimicrob Agents Chemother. 2015 Oct 19;60(1):151-60.