Difference between revisions of "Part:BBa K2933130"

 
Line 17: Line 17:
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K2933130 parameters</partinfo>
 
<partinfo>BBa_K2933130 parameters</partinfo>
 +
<!-- -->
 +
===Usage and Biology===
 +
 +
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.
 +
 +
<!-- -->
 +
<span class='h3bb'>Sequence and Features</span>
 +
<partinfo>BBa_K2933011 SequenceAndFeatures</partinfo>
 +
 +
 +
 +
==Experimental results==
 +
===Molecular cloning===
 +
<p style="text-align: center;">
 +
  [[File:GIM-2-PCR.jpeg|400px|]]        [[File:GIM-2-veri.jpeg|250px|]]<br>
 +
 +
'''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
 +
 +
 +
 +
<!-- Uncomment this to enable Functional Parameter display
 +
===Functional Parameters===
 +
<partinfo>BBa_K2933011 parameters</partinfo>
 
<!-- -->
 
<!-- -->

Revision as of 13:41, 24 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 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.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 731
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 731
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 731
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 731
  • 1000
    COMPATIBLE WITH RFC[1000]


Experimental results

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