Difference between revisions of "Part:BBa K2933212"
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<partinfo>BBa_K2933212 parameters</partinfo>
 
<partinfo>BBa_K2933212 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 GIM-2. It encodes a protein which is GIM-2 fused with His tag. The fusion protein is about 27.4 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of GIM-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 GIM-2. It encodes a protein which is GIM-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 27.4 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of GIM-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-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;">
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   [[File:GIM-2-PCR1.jpeg|400px|]]        [[File:GIM-2-veri.jpeg|250px|]]<br>
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   [[File:GIM-2-PCR.jpeg|400px|]] <br>
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'''Figure 1.''' The result of PCR <br>
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'''Figure 1.''' Left: The result of PCR, Right:The result of double enzyme digestion verification
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</p>
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==References==
 
==References==

Latest revision as of 14:03, 23 September 2019


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

This part consists of T7 promoter, RBS and protein coding sequence（His+Linker f+GIM-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
    Illegal PstI site found at 935
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 169
    Illegal PstI site found at 935
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 47
    Illegal PstI site found at 935
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 47
    Illegal PstI site found at 935
  • 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 GIM-2. It encodes a protein which is GIM-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 27.4 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of GIM-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.

GIM-2-PCR.jpeg
Figure 1. The result of PCR

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