Product Class: Competent Cell

Lemo21(DE3) Competent E. coli

No dry ice charges with competent cells
Catalog #SizeConcentration
C2528H6 x 0.05 ml/tube

Description

Highlights

  • Transformation efficiency: 1–3 x 107 cfu/µg pUC19 DNA
  • BL21(DE3) containing the Lemo System™
  • Tunable T7 Expression Strain for difficult targets: membrane proteins, toxic proteins and proteins prone to insoluble expression
  • Deficient in proteases Lon and OmpT
  • Resistant to phage T1 (fhuA2)
  • Lemo System™ maintained by chloramphenicol
Chemically competent E. coli cells suitable for transformation and protein expression. Fine tuning of T7 expression can alleviate inclusion body formation or growth inhibitory effects from toxic proteins. In many cases, less expression equals more protein of interest produced in the desired form. This is particularly true for membrane protein expression (Wagner et al. 2008). Membrane protein expression and protein export in E. coli are both limited by the throughput capacity of the Sec translocase and in some cases the Tat translocase. T7 expression of proteins targeted to the Sec translocase often leads to accumulation of inclusion bodies or inhibition of cell division if expression is not regulated. 

Lemo21(DE3) offers the host features of BL21(DE3) while also allowing for tunable expression of difficult clones. Tunable expression is achieved by varying the level of lysozyme (lysY), the natural inhibitor of T7 RNA polymerase. The level of lysozyme is modulated by adding L-rhamnose to the expression culture at levels from zero to 2000 µM. When Lemo21(DE3) is grown without rhamnose, the strain performs the same as a pLysS containing strain. However, optional addition of rhamnose tunes the expression of the protein of interest. For difficult soluble proteins, tuning the expression level may also result in more soluble, properly folded protein.

Features

  • Tunable T7 expression for difficult targets

Genotype

fhuA2 [lon] ompT gal (λ DE3) [dcm] ∆hsdS/ pLemo(CamR)
 λ DE3 = λ sBamHIo ∆EcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 ∆nin5
pLemo = pACYC184-PrhaBAD-lysY

Reagents Supplied

The following reagents are supplied with this product:

Store at (°C)Concentration
L-rhamnose solution500 mM
pUC19 Transformation Control Plasmid-200.05 ng/μl

Advantages and Features

Applications


Western analysis of 6-His tagged Brugia malayi protein. A) B. malayi protein expressed at 20°C in BL21(DE3). B) Soluble fractions of B. malayi protein expressed at 30°C.

Overnight expression of a membrane protein – PhoA fusion: Lemo System™ enables simple, rapid optimization of membrane protein expression.

Properties and Usage

Antibiotics for Plasmid SelectionWorking Concentration
Ampicillin100 μg/ml
Carbenicillin100 μg/ml
Kanamycin30 μg/ml
Streptomycin25 μg/ml
Tetracycline15 μg/ml

Storage Temperature

-80°C

Shipping Notes

  • Ships on dry ice

Quality Control

Quality Control Assays

The following Quality Control Tests are performed on each new lot and meet the specifications designated for the product. Individual lot data can be found on the Product Summary Sheet/Datacard or Manual which can be found in the Supporting Documents section of this page.
  • Transformation Efficiency:
    The competent cells are tested for transformation efficiency and pass minimum release criteria. Transformation efficiency is defined as the number of colony forming units (cfu) which would be produced by transforming 1 μg of plasmid into a given volume of competent cells.

Supporting Documents

Material Safety Datasheets

The following is a list of Material Safety Data Sheets (MSDS) that apply to this product to help you use it safely. The following file naming structure is used to name these document files: [Product Name] MSDS. For international versions please contact us at info@neb.com.

Datacards

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

Notes

  1. CAUTION: This product contains DMSO, a hazardous material. Review the MSDS before handling.
  2. STORAGE AND HANDLING: Competent cells should be stored at -80°C. Storage at -20°C will result in a significant decrease in transformation efficiency. Cells lose efficiency whenever they are warmed above -80°C, even if they do not thaw.
  3. Chloramphenicol must be added at 30 μg/ml to maintain the Lemo System™.
  1. What are the solutions/recipes (C2528)?
  2. What are the strain properties (C2528)?
  3. Why are there no colonies or no growth in liquid culture (C2529)?
  4. Why is induced protein insoluble (C2528)?
  5. Why is there no protein visible on gel or no activity (C2528)?
  6. Can I store competent cells at -20°C instead of -80°C?
  7. Which kind of transformation tubes should be used?
  8. What volume of DNA can be added into competent cells?
  9. What is the shelf life for this strain (NEB #C2528H)?
  10. Are NEB's competent cells compatible with the "Plate and Go" protocol?
  1. 5 Minute Transformation Protocol (C2528)
  2. Protein Expression Using Lemo21(DE3) (C2528)
  3. Transformation Protocol (C2528)

Selection Tools

Usage Guidelines & Tips

Application Notes