BL21(λDE3)pBirA Chemically Competent E. coli Cells for in vivo Biotinylation

The BL21(λDE3)pBirA strain is highly recommended for the high-efficiency expression and in vivo biotinylation of target proteins fused with an AviTag™ peptide or other specific peptide sequences recognized by E. coli BirA biotin ligase.

This chemically competent E. coli strain features an IPTG-inducible BirA expression plasmid carrying a streptomycin/spectinomycin resistance gene. It enables high-yield protein expression of any gene under the control of a T7 or E. coli promoter equipped with a ribosome binding site. The pBirA plasmid is fully compatible for co-expression with popular E. coli vectors, including pET and pTrc.

Key Applications & Usage

After transforming the BL21(λDE3)pBirA chemically competent cells with your target vector (carrying an AviTag™ sequence and an antibiotic resistance gene other than streptomycin/spectinomycin), grow the culture in the presence of both selection antibiotics. Induce BirA expression using IPTG and add exogenous d-biotin to the media at the time of induction at a final concentration of 50 µM to 200 µM.

Suggested Transformation Procedure

1. Thaw Cells: Thaw competent cells on ice, mix gently, and aliquot 50–100 µL into chilled tubes.
2. Add DNA: Add ≤5 µL plasmid DNA per 50 µL of cells; swirl gently to mix.
3. Ice Incubation: Incubate on ice for 30 minutes.
4. Heat Shock: Heat shock the cells in a water bath at 42°C for 30–45 seconds, then place back on ice for 2 minutes.
5. Recovery: Add 150 µL of SOC medium (optimal for max recovery).
6. Outgrowth: Shake at 200 rpm for 1 hour at 37°C.
7. Plating: Plate 100–200 µL onto LB agar plates containing the appropriate selection antibiotics; incubate overnight at 37°C.

Optimized Induction Protocol

1. Overnight Culture: Inoculate 5 mL of LB medium (containing 50 µg/mL streptomycin and target vector selection antibiotics) with single colonies; shake at 37°C overnight.
2. Subculture: Scale up into 1 L of LB medium; grow at 37°C until the OD600 reaches 0.6.
3. Induction: Induce protein expression with IPTG (1 mM final concentration) and add d-biotin (50 µM).*
4. Expression: Incubate at 30°C with shaking for 4–6 hours.
5. Harvest: Collect 20 µL samples of induced vs. non-induced cultures at 2, 4, and 6 hours for SDS-PAGE analysis. Harvest the remaining cell pellet via centrifugation at 6,000 × g for 10 minutes.

*Note: Optimal values for IPTG, biotin concentrations, and induction times may vary and should be optimized depending on the specific gene being expressed.

Product Documentation

For step-by-step instructions and technical guidance, download the formal BL21(λDE3)pBirA Product Manual (PDF).

Product Specifications

• Catalog #: BLB-201
• Transformation Efficiency: ≥ 1x10^6 cfu/µg pBR322 plasmid DNA
• Genotype: fhuA2 [lon] ompT gal (λ DE3) [dcm] ∆hsdS pBirA(StrR) [λ DE3 = λ sBamHIo ∆EcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 ∆nin5]
• Storage Conditions: Store at -80°C

Intended Use: For laboratory research use only (RUO).