Difference between revisions of "Part:BBa K2933011"
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===Exploration of expression condition===
 
===Exploration of expression condition===
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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 ampicillin(100 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 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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'''Purification of GST fusion proteins:'''<br>
 
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Revision as of 11:36, 6 September 2019


subclass B1 metallo-beta-lactamase GIM-2, codon optimized in E. coli

This part encodes a protein called GIM-2, which is a metallo-beta-lactamase of subclass B1.


Usage and Biology

GIM-2 is a new variant of GIM-1 with a single mutation, A290G, which was recently discovered. The German imipenemase-1 (GIM-1) MBL was first identified in clinical isolates of Pseudomonas aeruginosa in Germany in 2002. Recently, GIM-1 has been identified in other bacterial species, such as Serratia marcescens, Enterobacter cloacae, and Acinetobacter pittii , indicating that it is transmitted on mobile genetic elements. As a typical type of metallo-beta-lactamases which make bacteria antibiotic-resistant, it can hydrolyze extensive substrate and may pose a threat to human life and health.


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

Exploration of expression condition

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