Difference between revisions of "Part:BBa K2933217"
(→‎Usage and Biology)
 
(5 intermediate revisions by the same user not shown)
Line 18: Line 18:
 
<partinfo>BBa_K2933217 parameters</partinfo>
 
<partinfo>BBa_K2933217 parameters</partinfo>
 
<!-- -->
 
<!-- -->
−
==Usage and Biology===
+
==Usage and Biology==
−
This composite part is made up with three basic parts, the His tag, T7 promoter, RBS and our target protein VIM-66. It encodes a protein which is VIM-66 fused with His tag. The fusion protein is about 28.3 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of VIM-66 .  It is convenient for us to purify our target protein.<br>
+
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 VIM-66. It encodes a protein which is VIM-66 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 28.3 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of VIM-66 .  It is convenient for us to purify our target protein.<br>
 
===Molecular cloning===
 
===Molecular cloning===
 
We insert VIM-66 gene into the standard vector then transfer it into E.coli.
 
We insert VIM-66 gene into the standard vector then transfer it into E.coli.
−
   [[File:VIM-66-PCR.jpeg|600px|center|]]   
+
   [[File:VIM-66-PCR1.jpeg|600px|center|]]   
 
<p style="text-align: center;">
 
<p style="text-align: center;">
−
'''Figure 1.''' Left: The result of PCR, Right:The result of double enzyme digestion verification.LaneM,Marker, Lane1, the plasmid with VIM-66, Lane2, after double enzyme verification
+
'''Figure 1.''' The result of PCR
 +
 
 +
==References==
 +
1. Yoshihiro Yamaguchi. Wanchun Jin. Kazuyo Matsunaga. Crystallographic investigation of the inhibition mode of a VIM-2 metallo-beta-lactamase from Pseudomonas aeruginosa by a mercaptocarboxylate inhibitor. J. Med. Chem.200750266647-6653
 +
 
 +
2. Biochemical, Mechanistic, and Spectroscopic Characterizationof Metallo-β-lactamase VIM‑2[J]. Biochemistry, 2014, 53(46):7321-7331.
 +
 
 +
3. Christopeit T , Carlsen T J , Helland R , et al. Discovery of novel inhibitor scaffolds against the metallo-β-lactamase VIM-2 by SPR based fragment screening[J]. Journal of Medicinal Chemistry, 2015:151017114758002.
 +
 
 +
4. Christopeit T , Yang K W , Yang S K , et al. The structure of the metallo-β-lactamase VIM-2 in complex with a triazolylthioacetamide inhibitor[J]. 2016.

Latest revision as of 14:04, 23 September 2019


T7 promoter+RBS b+Linker h+His+Linker f+VIM-66+T7 terminator

This part consists of T7 promoter, RBS and protein coding sequence（His+Linker f+VIM-66），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
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 169
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 942
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 47
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 47
  • 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 VIM-66. It encodes a protein which is VIM-66 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 28.3 kD. In order to gain the highly purified target protein, we add His tag in N-terminal of VIM-66 . It is convenient for us to purify our target protein.

Molecular cloning

We insert VIM-66 gene into the standard vector then transfer it into E.coli.

VIM-66-PCR1.jpeg

Figure 1. The result of PCR

References

1. Yoshihiro Yamaguchi. Wanchun Jin. Kazuyo Matsunaga. Crystallographic investigation of the inhibition mode of a VIM-2 metallo-beta-lactamase from Pseudomonas aeruginosa by a mercaptocarboxylate inhibitor. J. Med. Chem.200750266647-6653

2. Biochemical, Mechanistic, and Spectroscopic Characterizationof Metallo-β-lactamase VIM‑2[J]. Biochemistry, 2014, 53(46):7321-7331.

3. Christopeit T , Carlsen T J , Helland R , et al. Discovery of novel inhibitor scaffolds against the metallo-β-lactamase VIM-2 by SPR based fragment screening[J]. Journal of Medicinal Chemistry, 2015:151017114758002.

4. Christopeit T , Yang K W , Yang S K , et al. The structure of the metallo-β-lactamase VIM-2 in complex with a triazolylthioacetamide inhibitor[J]. 2016.