Difference between revisions of "Part:BBa K2933170"

 
 
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<partinfo>BBa_K2933170 parameters</partinfo>
 
<partinfo>BBa_K2933170 parameters</partinfo>
 
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===Usage and Biology===
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This composite part is made up with six basic parts(Tac promoter，RBS a ,  Linker g, GST, Linker e and GIM-2). It encodes a protein which is GIM-2 fused with GST tag. The fusion protein is about 53.4 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of GIM-2 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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===Molecular cloning===
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First, we used the vector pGEX-6p 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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<p style="text-align: center;">
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  [[File:GIM-2-PCR.jpeg|400px|]]        [[File:GIM-2-veri.jpeg|250px|]]<br>
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'''Figure 1.''' Left: The result of PCR, Right:The result of double enzyme digestion verification
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===References===
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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.
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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.
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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.
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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

Latest revision as of 02:01, 26 September 2019


Tac promoter+RBS a+Linker g+GST+Linker e+GIM-2

This part consists of Tac promoter,RBS and protein coding sequence (GST+Linker e+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 PstI site found at 1525
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 1525
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 1525
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 1525
  • 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 GIM-2). It encodes a protein which is GIM-2 fused with GST tag. The fusion protein is about 53.4 kD. In order to gain the highly purified target protein, we add GST tag in N-terminal of GIM-2 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 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 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