Difference between revisions of "Part:BBa K2933016"
 
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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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  [[File:VIM-66-PCR.jpeg|600px|center|]] 
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<p style="text-align: center;">
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'''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
  
  
 
===Exploration of expression condition===
 
===Exploration of expression condition===
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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===
 
===Expression and purification===
 
'''Pre-expression:'''<br>
 
'''Pre-expression:'''<br>
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The bacteria were cultured in 5mL LB liquid medium with ampicillin(100 μg/mL final concentration) in 37℃ overnight.<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>
  
 
'''Massive expressing:'''<br>
 
'''Massive expressing:'''<br>
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After taking samples, we transfer them into 1L LB medium and add antibiotic to 100 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 0.3 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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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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'''Purification of GST fusion proteins:'''<br>
 
  
  
  
  
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==References==
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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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[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.
+
[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.
+
[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.
+
[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 11:12, 24 September 2019


subclass B1 metallo-beta-lactamase VIM-66, codon optimized in E. coli

This part encodes a protein called VIM-66, which is a metallo-beta-lactamase of subclass B1.


Usage and Biology

Metallo-beta-lactamase VIM was first identified in a patient in Italy who was infected with P. aeruginosa. 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.

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. VIM-expressing bacteria have been shown resistant to an array of β-lactam-containing antibiotics.

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.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 738
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Molecular cloning

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

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

Exploration of expression condition

VIM-28a-1.jpeg






Figure 2. The result of SDS-PAGE.
Lane1, uninduced VIM-66 with His tag(BBa_K2933155)(28.3kD).
Lane2, 37°C induced VIM-66 with His tag(28.3kD)
with 0.5mM IPTG.



Expression and purification

Pre-expression:
The bacteria were cultured in 5mL LB liquid medium with kanamycin(50 μg/mL final concentration) in 37℃ overnight.

Massive expressing:
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.



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.