Product Class: Other

SHuffle® T7 Competent E. coli

NEB recommends using SHuffle Express strains for best performance.

Product Introduction

Chemically competent E. coli K12 cells suitable for T7 protein expression with enhanced capacity to correctly fold proteins with multiple disulfide bonds in the cytoplasm.

  • T7 expression
  • Engineered E. coli K12 to promote disulfide bond formation in the cytoplasm
  • Resistant to phage T1 (fhuA2)
  • Free of animal products
  • NEB does not add a dry ice surcharge to our competent cell shipments
Catalog # Size Concentration
C3026J 12.0 x 0.05 ml

Product Information

Description

Highlights

  • Engineered E. coli K12 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)
  • Expresses a chromosomal copy of T7 RNAP
  • Tight control of expression by lacIq allows potentially toxic genes to be cloned
  • Resistance to phage T1 (fhuA2), Nit, Str, Spec

Transformation Efficiency

1 x 106 cfu/µg pUC19 DNA Chemically competent E. coli K12 cells engineered to form proteins containing disulfide bonds in the cytoplasm. Suitable for T7 promoter driven protein expression.

Genotype

F´ lac, pro, lacIq / Δ(ara-leu)7697 araD139 fhuA2 lacZ::T7 gene1 Δ(phoA)PvuII phoR ahpC* galE (or U) galK λatt::pNEB3-r1-cDsbC (SpecR, lacIq) ΔtrxB rpsL150(StrR) Δgor Δ(malF)3
This product is related to the following categories:
SHuffle Expression,
E. coli Protein Expression Strains Products,
Protein Expression Products,
E. coli Expression Strains Products,
This product can be used in the following applications:
T7 Expression,
Expression of Difficult Proteins,
Disulfide-bonded Protein Expression,
Protein Expression in E. Coli,
Protein Expression

Reagents Supplied

Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration

Properties & Usage

Antibiotic for Plasmid Selection

Antibiotics for Plasmid Selection Working Concentration
Ampicillin 100 µg/ml
Carbenicillin 100 µg/ml
Chloramphenicol 33 µg/ml
Kanamycin 30 µg/ml
Tetracycline 15 µg/ml

Shipping Notes

  • Ships on dry ice

Antibiotic Resistance

  • str
  • nit
  • spec

Features

  • T7 expression/K12 strain
  • Enhanced capacity to correctly fold proteins with multiple disulfide bonds in the cytoplasm

Application Features


Figure 1, vtPA activity assayed from crude lysates: 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: 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.

Product Notes

  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.

References

  1. Bessette, P.H. et al. (1999). Proc. Natl. Acad. Sci. USA. 96, 13703-13708.
  2. Qiu, J., Swartz, J.R. and Georgiou, G. (1998). 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.

Protocols, Manuals & Usage

Protocols

  1. Expression Using SHuffle (C3026)
  2. Transformation Protocol (#C3029, #C3026, #C3028 and #C3030)

Usage & Guidelines

Application Notes

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  1. What are the strain properties (C3026)?
  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. Does Streptomycin and/or Spectinomycin need to be added when growing 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?
  16. Is the genome of SHuffle strains available?
  17. Is T7 expression subject to catabolite repression in NEB T7 Express or SHuffle strains?
  18. Can one use SHuffle cells to secrete my protein of interest?

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

Licenses

The buyer and user have a non-exclusive sub-license to use this system or any component thereof for RESEARCH PURPOSES ONLY, based upon agreement to the following assurances.

Transfer of the host cells that contain the cloned copy of the T7 gene 1 to third parties is explicitly prohibited. This limitation applies to E. coli ER2566, ER2833, ER3011, ER3012, ER3013 and ER3021, SHuffle T7, SHuffle T7 LysY, SHuffle T7 Express, SHuffle T7 Express LysY and their competent derivatives, C2566, C2833, C3010, C3013, C3016, C3022, C3026, C3027, C3029 and C3030 when provided separately or when provided in combination with appropriate vectors for said systems.

A license to use this system or any components thereof for commercial purposes may be obtained from New England Biolabs, Inc.

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