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  • SHuffle® T7 Express Competent E. coli


    Chemically competent E. coli B cells engineered to form proteins containing disulfide bands in the cytoplasm. Suitable for T7 promoter driven protein expression.


    • Transformation efficiency: 1 x 107 cfu/µg pUC19 DNA
    • Engineered E. coli B strain to promote disulfide bond formation in the cytoplasm
    • Constitutively expresses a chromosomal copy of the disufide bond isomerase DsbC
    • DsbC promotes the correction of mis-oxidized proteins into their correct form (1,3)
    • The cytoplasmic DsbC is also a chaperone that can assist in the folding of proteins that do not require disulfide bonds (4)
    • Enhanced BL21 derivative
    • Expresses a chromosomal copy of T7 RNAP
    • Activity of nonspecific endonuclease I (endA1) eliminated for highest quality plasmid preparations
    • Deficient in proteases Lon and OmpT
    • Resistance to phage T1 (fhuA2)


    fhuA2 lacZ::T7 gene1 [lon] ompT ahpC gal λatt::pNEB3-r1-cDsbC (SpecR, lacIq) ΔtrxB sulA11 R(mcr-73::miniTn10--TetS)2 [dcm] R(zgb-210::Tn10 --TetS) endA1 Δgor ∆(mcrC-mrr)114::IS10

    Reagents Supplied

    The following reagents are supplied with this product:

    Store at (°C)Concentration
    SHuffle Competent E. coli-80

    Advantages and Features


    • T7 expression
    • Protease deficient/B strain
    • Enhanced capacity to correctly fold proteins with multiple disulfide bonds in the cytoplasm


    Figure 1, vtPA activity assayed from crude lysates: Truncated tissue plasminogen activator (vtPA), which contains nine disulfide bonds when folded and oxidized correctly, was expressed from a pTrc99a plasmid in the cytoplasm of E. coli cells. After induction, cells were harvested and crude cell lysates were prepared. vtPA was assayed using a chromogenic substrate Chromozym t-PA (Roche #11093037001) and standardized to protein concentration using Bradford reagent. E. coli wt+ cells are DHB4, which is the parent of FÅ113 (Origami™).
    Figure 2, PfCHT1 chitinase activity assayed from crude lysates: Plasmodium falciparum chitinase (PfCHT1) with three cysteines were expressed from a plasmid under the regulation of T7 promoter. After induction, cells were harvested and crude cell lysates were prepared. PfCHT1 was assayed using a chromogenic substrate (CalBioChem #474550) and standardized to protein concentration using Bradford reagent.

    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


    Shipping Notes

    • Ships on dry ice

    Antibiotic Resistance

    • spec⁽⁷⁾

    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.
    • Functional Test (Disulfide Bond Formation):
      Cells are transformed with a plasmid expressing MBP-NucA fusion protein. When expressed  at 37° C in E. coli, NucA is toxic to cells only in its oxidized disulfide-bonded state.
    • 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.


    1. 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.


    1. Bessette, P.H. et al. (1999). Proc. Natl. Acad. Sci. USA. 96, 13703-13708.
    2. Qiu, J., Swartz, J.R. and Georgiou, G. (1999). Appl. Environ. Microbiol. 64, 4891-4896.
    3. Levy, R. et al. (2001). Protein Expr. Purif. 23, 338-347.
    4. Chen, J. et al. (1999). J. Biol. Chem. 274, 19601-19605.
    5. Boyd, D. et al. (2000). J. Bacteriol. 182, 842-847.
    6. de Marco, A. (2009). Microbial Cell Factories. 8, 26.

    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.


    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. What are the strain properties (C3029)?
    2. What applications are SHuffle® strains useful for?
    3. Does my protein have disulfide bonds?
    4. Which SHuffle® strain should I use?
    5. Is there anything I can do to increase protein yield when using SHuffle strains?
    6. What are the growth characteristics of the SHuffle strains?
    7. How do SHuffle® strains aid in cytoplasmic disulfide bond formation?
    8. Can I store competent cells at -20°C instead of -80°C?
    9. How should I express my protein of interest in SHuffle?
    10. Do Streptomycin and/or Spectinomycin need to be added to SHuffle strains?
    11. Can I conduct M13 phage display experiments in SHuffle strains?
    12. Can I conduct blue/white screening using alpha complementation of lacZ in SHuffle strains?
    13. Which SHuffle strains are resistant to chloramphenicol? Is chloramphenicol required for maintenance of the mini F plasmid?
    14. Do SHuffle cells grow in minimal media?
    15. Are SHuffle strains temperature sensitive?
    1. 5 Minute Transformation Protocol (C3029)
    2. Expression Using SHuffle (C3029)
    3. High Efficiency Transformation Protocol

    Selection Tools

    Usage Guidelines & Tips

    Application Notes