BL21 (DE3) Chemically Competent E. coli Cells

GoldBio’s BL21 (DE3) Chemically Competent E. coli cells are are high efficiency cells, suitable for a wide variety of applications such as transformation and routine protein expression. BL21 (DE3) chemically competent cells feature a widely used host background, a T7 expression strain, and are deficient in both Ion (1) and ompT proteases. In addition, BL21 (DE3) cells are resistant to phage T1 (fhuA2) and are B strain.

BL21 (DES) chemically competent cells are free of animal-derived products and grown with animal-free media.

BL21 (DE3) Highlights

• Free of all Animal products
• High plasmid stability and decreased plasmid transfer
• No DNA cytosine methylation means better restriction digestion and sequencing
• Galactose deficiency makes it ideal for selective systems
• Carries IPTG-inducible T7 RNA polymerase for high-level expression

Kit Components

• Competent Cells
• 1 x 12 mL Recovery Media
• 1 x 10 µL Control Plasmid (pUC19 Control, 500 pg/µL)

Reagents Needed for One Reaction

• BL21 (DE3) chemically competent cells: 50 µL
• DNA (or pUC19 Control, 500 pg/µL): 2 µL
• Recovery medium: 1 mL

Storage/Handling

This product may be shipped on dry ice. BL21 Chemically Competent E. coli cells should be stored at -80°C, pUC19 Control DNA should be stored at -20°C and recovery medium should be stored at 4°C immediately upon arrival. When stored under the recommended conditions and handled correctly, these products should be stable for at least 1 year from the date of receipt.

Genomic Features

• ≥5 x 10⁵ cfu/µg efficiency
• Widely used host background
• T7 Expression Strain
• Deficient in both lon (1) and ompT proteases.
• Resistant to phage T1 (fhuA2).
• B Strain

Genotype

F–ompT hsdS(rB– mB–) gal dcm λ(DE3)

Applications

• High-level recombinant protein expression (especially with T7 promoter-based systems like pET vectors)
• Expression of toxic proteins with tight IPTG-inducible control
• Purification of His-tagged proteins using Ni-NTA affinity chromatography
• Protein expression for crystallography or structural studies
• In vitro transcription studies using T7 RNA polymerase
• Expression of fusion proteins (e.g., GST, MBP, or fluorescent tags)

Quality Control

Transformation efficiency is tested by using the pUC19 control DNA supplied with the kit and using the protocol given below. Transformation efficiency should be ≥5 x 10⁵ CFU/µg pUC19 DNA. Untransformed cells are tested for appropriate antibiotic sensitivity.

General Guidelines

• Handle competent cells gently as they are highly sensitive to changes in temperature or mechanical lysis caused by pipetting.
• Thaw competent cells on ice and transform cells immediately following thawing. After adding DNA, mix by tapping the tube gently. Do not mix cells by pipetting or vortexing.

Calculation of Transformation Efficiency

Transformation Efficiency (TE) is defined as the number of colony forming units (cfu) produced by transforming 1 µg of plasmid into a given volume of competent cells.

• TE = Colonies/µg/Dilution
  • Colonies = the number of colonies counted
  • µg = amount of DNA transformed in µg
  • Dilution = total dilution of the DNA before plating

Example: Transform 1 µl of (10 pg/µl) control plasmid into 25 µl of cells, add 975 µl of Recovery Medium. Dilute 10 µl of this in 990 µl of Recovery Medium and plate 50 µl. Count the colonies on the plate the next day. If you count 250 colonies, the TE is calculated as follows:

Colonies = 250
µg of DNA = 0.00001
Dilution = 10/1000 x 50/1000 = 0.0005
TE = 250/0.00001/0.0005 = 5.0 × 10¹⁰