Difference between revisions of "Part:BBa K2933130"

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===Usage and Biology===
 
===Usage and Biology===
  
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This composite part is made up with four basic parts, the His tag, the Sumo tag, three cutting sites(NdeI, NheI and BamHI) and our target protein GIM-2. It encodes a protein which is GIM-2 fused with His-Sumo tag. The fusion protein is about 39.4kD. In order to gain the highly purified target protein, we add His-Sumo tag in N-terminal of GIM-2 and combine the three parts with these three cutting sites. The fusion protein can be cut off at the cutting site BamHI. It is convenient for us to purify our target protein.
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This composite part is made up with five basic parts, the His tag, the Sumo tag, three cutting sites(NdeI, NheI and BamHI) and our target protein GIM-2. It encodes a protein which is GIM-2 fused with His-Sumo tag. The fusion protein is about 39.4kD. In order to gain the highly purified target protein, we add His-Sumo tag in N-terminal of GIM-2 and combine the three parts with these three cutting sites. The fusion protein can be cut off at the cutting site BamHI. It is convenient for us to purify our target protein.
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===Molecular cloning===
 
===Molecular cloning===

Revision as of 11:36, 25 September 2019


His+Linker a+Sumo+Linker b+GIM-2

This part encodes the fusion protein of His tag, sumo tag and GIM-2 to promote the expression and purification of target protein(GIM-2).


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 256
    Illegal PstI site found at 1086
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 256
    Illegal NheI site found at 33
    Illegal PstI site found at 1086
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 256
    Illegal BglII site found at 145
    Illegal BamHI site found at 344
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 256
    Illegal PstI site found at 1086
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 256
    Illegal PstI site found at 1086
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This composite part is made up with five basic parts, the His tag, the Sumo tag, three cutting sites(NdeI, NheI and BamHI) and our target protein GIM-2. It encodes a protein which is GIM-2 fused with His-Sumo tag. The fusion protein is about 39.4kD. In order to gain the highly purified target protein, we add His-Sumo tag in N-terminal of GIM-2 and combine the three parts with these three cutting sites. The fusion protein can be cut off at the cutting site BamHI. It is convenient for us to purify our target protein.

Molecular cloning

GIM-2-PCR.jpeg GIM-2-veri.jpeg
Figure 1. Left: The result of PCR, Right:The result of double enzyme digestion verification

References

[1]Skagseth S , Akhter S , Paulsen M H , et al. Metallo-β-lactamase inhibitors by bioisosteric replacement: Preparation, activity and binding[J]. European Journal of Medicinal Chemistry, 2017, 135:159-173.

[2]Wendel AF, MacKenzie CR. 2015. Characterization of a novel metallo-lactamase variant, GIM-2, from a clinical isolate of Enterobacter cloacae in Germany. Antimicrob Agents Chemother 59:1824 –1825.

[3]Borra P S , Samuelsen O , Spencer J , et al. Crystal Structures of Pseudomonas aeruginosa GIM-1: Active-Site Plasticity in Metallo-beta-Lactamases[J]. Antimicrobial Agents and Chemotherapy, 2013, 57(2):848-854.

[4]Susann S, Trine J C, Gro Elin K B, James S, Ørjan S, Hanna-Kirsti S. L. Role of Residues W228 and Y233 in the Structure and Activity of Metallo-β-Lactamase GIM-1. Antimicrobial Agents and Chemotherapy Jan 2016, 60 (2) 990-1002