Difference between revisions of "Part:BBa K2933142"
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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>
 
After verification, it was determined that the construction is successful. We converted the plasmid to ''E. coli'' BL21(DE3) for expression and purification.<br>
 +
===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.

Revision as of 12:44, 22 September 2019


His+Linker f+SPG-1

This part encodes the fusion protein of His tag and SPG-1 to promote the expression and purification of target protein(SPG-1).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 51
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This composite part is made up with three basic parts, the HiS tag, the thrombin restriction site 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. It is convenient for us to purify our target protein.

Molecular cloning

First, we used the vector pET28a 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.

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.