pBAD.Sigma32.I54N
(Plasmid #59982)

• Purpose: Expresses sigma32 I54N mutant in E. coli to render a heat-shock like cellular folding environment
• Depositing Lab: Jeffery Kelly
• Publication: Zhang et al ACS Chem Biol. 2014 Jul 22.

Backbone

• Vector backbone: pBAD
• Backbone size w/o insert (bp): 4000
• Total vector size (bp): 5000
• Modifications to backbone: n/a
• Vector type: Bacterial Expression

Growth in Bacteria

• Bacterial Resistance(s): Ampicillin, 100 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): BL21DE3
• Growth instructions: Recombinant protein over-expression in the heat-shock-like expression system. 1. A pET29b(+) vector (kanamycin resistance) encoding the gene of a protein-of-interest (POI) was transformed into the Bl21 (DE3) strain harboring the pBAD-σ32 (I54N) vector (ampicillin resistance). 2. The Bl21 (DE3) cells bearing both vectors were selected on LB-agar plates (kanamycin and ampicillin) and a single colony was grown in 15 mL LB media supplemented with kanamycin and ampicillin overnight at 37 ºC. 3. The overnight culture was then used to inoculate a culture in a larger volume at 37 ºC, and cell growth was monitored by measuring the optical density (OD) at 600 nm (OD600) using a UV-Vis spectrometer. 4. When the OD600 reached 0.4, σ32-I54N expression was induced with L-arabinose, whose concentration can be adjusted between 0.02 - 0.2% (w/v) to optimize the desired condition. 5. An enhanced E. coli proteostasis capacity was achieved within 1 h, and isopropyl b-D-1-thiogalactopyranoside (IPTG, final concentration of 1mM) was then added to induce over-expression of the POI, which could be induced for desired durations as long as 24 h. 6. During POI expression, L-arabinose was kept in the cell culture to ensure that the E. coli proteostasis network capacity was constantly enhanced. 7. For induction period longer than 16 hours, it is recommended that ampicillin be replaced by carbenicillin because the latter is more resistant than ampicillin to degradation by b-lactamase enzymes.
• Copy number: Low Copy

Gene/Insert

• Gene/Insert name: Sigma32 I54N
• Species: E. coli
• Mutation: I54N
• GenBank ID: unknown unknown

Resource Information

• A portion of this plasmid was derived from a plasmid made by: Dr. Xin Zhang cloned this gene at The Scripps Research Institute
• Article Citing this Plasmid:
  • 1 Reference

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
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