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  • NiCo21(DE3) Competent E. coli

    Description

    Chemically competent E. coli cells derived from BL21(DE3). Poly-histidine tagged recombinant proteins that are isolated by immobilized metal affinity chromatography (IMAC) are often contaminated with significant amounts of endogenous E. coli metal binding proteins. The protein expression strain NiCo21(DE3) has been engineered to minimize E. coli protein contamination of IMAC fractions: GlmS is mutated to eliminate binding to IMAC resins and three other proteins (SlyD, ArnA and Can) are tagged to enable rapid removal by chitin affinity chromatography.

    Highlights

    • Superior alternative to BL21(DE3) for routine protein expression
    • Improved purity of target proteins isolated by IMAC
    • Identical growth characteristics as BL21(DE3)
    • Transformation efficiency: 1–5 x 107 cfu/µg pUC19
    • Deficient in proteases Lon and OmpT
    • Resistant to phage T1 (fhuA2)
    • Free of animal products

    Genotype

    can::CBD fhuA2 [lon] ompT gal (λ DE3) [dcm] arnA::CBD slyD::CBD glmS6Ala ∆hsdS λ DE3 = λ sBamHIo ∆EcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 ∆nin5

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    pUC19 Transformation Control Plasmid-200.05 ng/μl
    SOC Outgrowth Medium41X

    Advantages and Features

    Features

    • Improved purity of target proteins isolated by IMAC

    Applications


    Improved purity of His-tagged proteins with NiCo21(DE3) A) Expression of Glutamyl tRNA Synthetase (6-His) in NiCo21(DE3) Competent E. coli followed by Ni-NTA purification. Eluent (E) from a Ni-NTA column was passed over a chitin column. The protein of interest elutes in the flow through (FT), while the CBD-tagged metal binding proteins remain bound (B) to the chitin resin (NEB #S6651S ). Purifications were performed according to manufacturers' recommended conditions. B) Contaminants ArnA, SlyD and Can are confirmed by Western blot using Anti-CBD Antibody (NEB #E8034S ).

    Two-step purification of target protein that has been expressed in the NiCo21(DE3) strain of E. coli

    Properties and Usage

    Antibiotics for Plasmid SelectionWorking Concentration
    Ampicillin100 μg/ml
    Carbenicillin100 μg/ml
    Chloramphenicol33 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.

    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.

    References

    1. Bolanos-Garcia and Davies (2006). Biochimica et Biophysica Acta. 1760, 1304-1313.
    2. Carine Robichon, Jianying Luo, Thomas B. Causey, Jack S. Benner, and James C. Samuelson (2011). Applied and Environmental Microbiology. 77, 4634-4646.

    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.
    1. Does New England Biolabs offer strains of Competent E.coli suited for protein expression?
    2. What are the solutions/recipes (C2529)?
    3. Why are there no colonies or no growth in liquid culture (C2529)?
    4. Why is the induced protein insoluble (C2529)?
    5. Why is there no protein visible by SDS-PAGE or no activity (C2529)?
    6. What are the strain properties (C2529)? 
    7. Can I store competent cells at -20°C instead of -80°C?
    8. Which kind of transformation tubes should be used?
    9. What volume of DNA can be added into competent cells?
    10. What is the shelf life for this strain (NEB #C2529H)?
    11. Are NEB's competent cells compatible with the "Plate and Go" protocol?
    1. 5 Minute Transformation Protocol (C2529)
    2. Expression Using NiCo21(DE3) (C2529)
    3. Protocol for Removal of IMAC Contaminating Proteins (C2529)
    4. High Efficiency Transformation Protocol (C2529)

    Selection Tools

    Usage Guidelines & Tips