Difference between revisions of "Part:BBa K2933162"

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===Usage and Biology===
 
===Usage and Biology===
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This composite part is made up with three basic parts(Tac promoter，RBS a , Linker g, GST, Linker e and SPG-1). It encodes a protein which is SPG-1 fused with GST tag. The fusion protein is about 56.2kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of SPG-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 six basic parts(Tac promoter，RBS a , Linker g, GST, Linker e and SPG-1). It encodes a protein which is SPG-1 fused with GST tag. The fusion protein is about 56.2kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of SPG-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>
  
 
===Molecular cloning===
 
===Molecular cloning===

Latest revision as of 13:39, 24 September 2019


Tac promoter+RBS a+Linker g+GST+Linker e+SPG-1

This part consists of Tac promoter,RBS and protein coding sequence (GST+Linker e+SPG-1),and the biological module can be built into E.coli for protein expression.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 181


Usage and Biology

This composite part is made up with six basic parts(Tac promoter，RBS a , Linker g, GST, Linker e and SPG-1). It encodes a protein which is SPG-1 fused with GST tag. The fusion protein is about 56.2kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of SPG-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.

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.

SPG-PCR.png
Figure 1. a: The PCR result of SPG. b: 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.

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.