Difference between revisions of "Part:BBa K2933258"
 
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===Usage and Biology===
 
===Usage and Biology===
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This composite part is made up with eight basic parts, T7 Ribosome binding sites,the His-Sumo tag, three cutting sites of Prescission Protease, our target protein VIM-66 and T7 terminator. It encodes a protein which is VIM-66 fused with His-Sumo tag. The fusion protein is about 40.3 kD. The fusion protein can be cut off at the cutting sites by Prescission Protease. It is convenient for us to purify our target protein.
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Metallo-beta-lactamase VIM was first identified in a patient in Italy who was infected with P. aeruginosa.
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VIM-66 sequence is mostly similar to VIM-2. Among these VIM variants, VIM-2 appears to be the one most commonly found in the clinic, and VIM-2-expressing bacterial strains have been found in many countries.
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In fact, blaVIM genes have been detected mostly in the Mediterranean countries of Europe, in the Far East regions, including Japan, and at present, in American regions, including the U.S.
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VIM-expressing bacteria have been shown resistant to an array of β-lactam-containing antibiotics.
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Due to the high clinical relevance, the widespread and the broad substrate range, VIM-2 is an important drug target to restore the function of carbapenem antibiotics and the treatment of antibiotic resistant bacteria.
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2933016 SequenceAndFeatures</partinfo>
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===Molecular cloning===
 
===Molecular cloning===
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We insert VIM-66 gene into the standard vector then transfer it into E.coli.
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we used the vector pET-28bs to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.
 
   [[File:VIM-66-PCR.jpeg|600px|center|]]   
 
   [[File:VIM-66-PCR.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.''' 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
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===References===
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[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
  
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[2]Biochemical, Mechanistic, and Spectroscopic Characterizationof Metallo-β-lactamase VIM‑2[J]. Biochemistry, 2014, 53(46):7321-7331.
  
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===Exploration of expression condition===
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[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.
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  [[File:VIM-28a-1.jpeg|160px|left|]] 
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'''Figure 2.'''
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The result of SDS-PAGE. <br>
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Lane1, uninduced VIM-66 with His tag(BBa_K2933155)(28.3kD). <br>
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Lane2, 37°C induced VIM-66 with His tag(28.3kD) <br>
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with 0.5mM IPTG.<br>
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===Expression and purification===
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'''Pre-expression:'''<br>
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The bacteria were cultured in 5mL LB liquid medium with kanamycin(50 μg/mL final concentration) in 37℃ overnight.<br>
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'''Massive expressing:'''<br>
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After taking samples, we transfer them into 1L LB medium and add antibiotic to 50 μg/mL final concentration. Grow them up in 37°C shaking incubator. Grow until an OD 600 nm of 0.8 to 1.2 (roughly 3-4 hours). Induce the culture to express protein by adding 0.5 mM IPTG (isopropylthiogalactoside, MW 238 g/mol) or ~0.1 gram per 1.5 liter flask. Put the liter flasks in 16°C shaking incubator for 16h. Centrifuge your bacteria in 500 mL bottles in the 4°C rotor at 4,000 rpm for 20 mins. Do this in batches until all your culture is spun down saving the cell pastes each time.<br>           
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==References==
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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
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2. Biochemical, Mechanistic, and Spectroscopic Characterizationof Metallo-β-lactamase VIM‑2[J]. Biochemistry, 2014, 53(46):7321-7331.
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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.
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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.
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[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 15:01, 25 September 2019


RBS b+Linker h+His+Linker a+Sumo+Linker b+VIM-66+T7 terminator

This part consists of RBS, protein coding sequence(His+Linker a+Sumo+Linker b+VIM-66) and T7 terminator,and the biological module can be build into E.coli for protein expression. This part can be prefaced with promoters of different strengths and types to regulate expression function.

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 298
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 298
    Illegal NheI site found at 75
    Illegal NheI site found at 1206
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 298
    Illegal BglII site found at 187
    Illegal BglII site found at 1135
    Illegal BamHI site found at 386
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 298
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 298
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

This composite part is made up with eight basic parts, T7 Ribosome binding sites,the His-Sumo tag, three cutting sites of Prescission Protease, our target protein VIM-66 and T7 terminator. It encodes a protein which is VIM-66 fused with His-Sumo tag. The fusion protein is about 40.3 kD. The fusion protein can be cut off at the cutting sites by Prescission Protease. It is convenient for us to purify our target protein.

Molecular cloning

we used the vector pET-28bs to construct our expression plasmid. And then we converted the plasmid constructed to E. coli DH5α to expand the plasmid largely.

VIM-66-PCR.jpeg

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

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.