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SHuffle® T7 Express Competent E. coli

Catalog # Concentration Size List Price Quantity Your Price
C3029J 12 x 0.05 ml $383.00
$344.70
Catalog # Size List Price Your Price
C3029J 12 x 0.05 ml $383.00
$344.70
Catalog #
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*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca

Chemically competent E. coli 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 B strain to promote disulfide bond formation in the cytoplasm
  • Deficient in proteases Lon and OmpT
  • Resistant to phage T1 (fhuA2)
  • Free of animal products
  • No dry ice charge to competent cell shipments
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  • Monarch_MiniprepProtocol_VideoThumb
    Stages of SHUFFLE® Optimization
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    Fast transformation plating with glass beads
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    What is a Disulfide Bond (1 of 4)

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

Highlights
  • 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), Nit, Spec, Str*

    *Resistance to low levels of streptomycin may be observed.
Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  SHuffle® T7 Express Competent E. coli C3029JVIAL -80 12 x 0.05 ml
Features
  • T7 expression
  • Protease deficient/B 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.

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 (resistance to low levels of streptomycin may be observed)
  • nit
  • spec


Companion Products
Notes
  • 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
  • Bessette, P.H. et al. (1999). Proc. Natl. Acad. Sci. USA. 96, 13703-13708.
  • Qiu, J., Swartz, J.R. and Georgiou, G. (1999). Appl. Environ. Microbiol. 64, 4891-4896.
  • Levy, R. et al. (2001). Protein Expr. Purif. 23, 338-347.
  • Chen, J. et al. (1999). J. Biol. Chem. 274, 19601-19605.
  • Boyd, D. et al. (2000). J. Bacteriol. 182, 842-847.
  • de Marco, A. (2009). Microbial Cell Factories. 8, 26.
Citations
Additional Citations
  • Agrawal, A., Bisharyan, Y., Papoyan, A, Bednenko, J., Cardarelli, J., Yao, M., Clark, T., Berkm​en, M., Ke, N., Colussi, P. (2019) Fusion to Tetrahymena thermophila granule lattice protein 1 confers solubility to sexual stage malaria antigens in Escherichia coli. Protein Expr Purif 153, 7-17.PubMedID: 30081196, DOI: 10.1016/j.pep.2018.08.001.
  • Robinson, M.-P., Ke, N., Lobstein, J., Peterson, C., Szkodny, A., Mansell, T.J., Tuckey, C., Riggs, P.D., Colussi, P.A., Noren, C.J., Taron, C.H., Delisa, M.P., Berkmen, M. (2015) Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria Nat Commun (6)8072,PubMedID: 26311203, DOI: 10.1038/ncomms9072.
  • Leith, E.M., O'Dell, W.B., Ke, N., McClung, C., Berkmen, M., Bergonzo, C., Brinson, R.G., Kelman, Z (2019) Characterization of the internal translation initiation region in monoclonal antibodies expressed in Escherichia coli J Biol Chem 294(48), 18046-18056..PubMedID: 31604819, DOI: 10.1074/jbc.RA119.011008
  • Anton, B.P., Fomenkov, A., Raleigh, E.A. and Berkmen, M. (2016) Complete Genome Sequence of the Engineered Escherichia coli SHuffle Strains and Their Wild-Type Parents Genome Announc Mar 31;4(2),PubMedID: 27034504, DOI: 10.1128/genomeA.00230-16.
  • Berkmen, M. (2012) Production of disulfide-bonded proteins in Escherichia coli Protein Expr Purif 240-251.PubMedID: 22085722
  • Reddy, P.T., Brinson, R.G., Hoopes, J.T., McClung, C., Ke, N., Kashi, L. (2018) Platform development for expression and purification of stable isotope labeled monoclonal antibodies in Escherichia coli. mAbs MAbs 10 (7), 992-1002.PubMedID: 30060704, DOI: 10.1080/19420862.2018.1496879
  • Ren, G., Ke, N. and Berkmen, M. (2016) Use of the Shuffle Strains in Production of Proteins. Curr Protoc Protein Sci Aug 1, 1;85:5.26.1-5.26.21..PubMedID: 27479507 , DOI: 10.1002/cpps.11.
Publications
  • Reddy, P.T., Brinson, R.G., Hoopes, J.T., McClung, C., Ke, N., Kashi, L. (2018). Platform development for expression and purification of stable isotope labeled monoclonal antibodies in Escherichia coli. mAbs MAbs. 10 (7), 992-1002.PubMedID: 30060704, DOI: 10.1080/19420862.2018.1496879
  • Agrawal, A., Bisharyan, Y., Papoyan, A, Bednenko, J., Cardarelli, J., Yao, M., Clark, T., Berkm​en, M., Ke, N., Colussi, P. (2019). Fusion to Tetrahymena thermophila granule lattice protein 1 confers solubility to sexual stage malaria antigens in Escherichia coli. Protein Expr Purif. 153, 7-17.PubMedID: 30081196, DOI: 10.1016/j.pep.2018.08.001.
  • Leith, E.M., O'Dell, W.B., Ke, N., McClung, C., Berkmen, M., Bergonzo, C., Brinson, R.G., Kelman, Z (2019). Characterization of the internal translation initiation region in monoclonal antibodies expressed in Escherichia coli J Biol Chem. 294(48), 18046-18056..PubMedID: 31604819, DOI: 10.1074/jbc.RA119.011008
  • Anton, B.P., Fomenkov, A., Raleigh, E.A. and Berkmen, M. (2016). Complete Genome Sequence of the Engineered Escherichia coli SHuffle Strains and Their Wild-Type Parents Genome Announc. Mar 31;4(2),PubMedID: 27034504, DOI: 10.1128/genomeA.00230-16.
  • Ren, G., Ke, N. and Berkmen, M. (2016). Use of the Shuffle Strains in Production of Proteins. Curr Protoc Protein Sci. Aug 1, 1;85:5.26.1-5.26.21..PubMedID: 27479507 , DOI: 10.1002/cpps.11.
  • Robinson, M.-P., Ke, N., Lobstein, J., Peterson, C., Szkodny, A., Mansell, T.J., Tuckey, C., Riggs, P.D., Colussi, P.A., Noren, C.J., Taron, C.H., Delisa, M.P., Berkmen, M. (2015). Efficient expression of full-length antibodies in the cytoplasm of engineered bacteria Nat Commun. (6)8072,PubMedID: 26311203, DOI: 10.1038/ncomms9072.
Quality Control Assay
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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 Data Sheets
Legal And Disclaimer

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.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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