Product Class: Other

Lemo21(DE3) Competent E. coli

Product Introduction

Chemically competent E. coli cells suitable for tunable T7 expression of challenging proteins.

  • 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)
  • Free of animal products
Bioz Badge Exists : True
Catalog # Size Concentration
C2528J 12 x 0.05 ml

Product Information

Description

Highlights

  • 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

Transformation Efficiency

1–3 x 107 cfu/µg pUC19 DNA 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.

Figure 1: Western analysis of 6-His tagged Brugia malayi protein

Gel showing expression of B. malayi 
A) B. malayi protein expressed at 20°C in BL21(DE3).
B) Soluble fractions of B. malayi protein expressed at 30°C.


Figure 2: Overnight expression of a membrane protein – PhoA fusion

Bar graph showing PhoA activity versus Rhamnose (µM) 

Lemo System™ enables simple, rapid optimization of membrane protein expression.

Genotype

fhuA2 [lon] ompT gal (λ DE3) [dcm] âˆ†hsdS/ pLemo(CamR)  λ DE3 = λ sBamHIo ∆EcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 ∆nin5 pLemo = pACYC184-PrhaBAD-lysY
This product is related to the following categories:
T7 Expression,
E. coli Protein Expression Strains,
E. coli Expression Strains,
Protein Expression,
This product can be used in the following applications:
T7 Expression,
Toxic Protein Expression,
Membrane Protein Expression,
Expression of Difficult Proteins,
Peptide Ligation,
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
  • NEB # Stored at (°C)
    C2528J -80
    Component Name Component # Stored at (°C) Amount Concentration
    pUC19 Vector N3041AVIAL -20 1 x 0.025 ml 50 pg/µl
    Lemo21(DE3) Competent E. coli C2528JVIAL -80 12 x 0.05 ml Not Applicable
    L-rhamnose solution B9030AVIAL -80 1 x 12.5 ml 0.5 M

Properties & Usage

Antibiotic for Plasmid Selection

Antibiotics for Plasmid Selection Working Concentration
Ampicillin 100 µg/ml
Carbenicillin 100 µg/ml
Kanamycin 30 µg/ml
Streptomycin 25 µg/ml
Tetracycline 15 µg/ml

Shipping Notes

  • Ships on dry ice

Antibiotic Resistance

  • cam

Features

  • Tunable T7 expression for difficult targets

Application Features



Figure 1: Western analysis of 6-His tagged Brugia malayi protein

Gel showing expression of B. malayi 
A) B. malayi protein expressed at 20°C in BL21(DE3).
B) Soluble fractions of B. malayi protein expressed at 30°C.


Figure 2: Overnight expression of a membrane protein – PhoA fusion

Bar graph showing PhoA activity versus Rhamnose (µM) 

Lemo System™ enables simple, rapid optimization of membrane protein expression.

Product 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™.

Tools & Resources

Selection Charts

FAQs & Troubleshooting

FAQs

  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 “Mix & Go" protocol?
  11. Is T7 expression subject to catabolite repression in BL21(DE3) and derivative strains?

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

Licenses/Patents/Disclaimers: 
The Lemo System™ (pACYC184-rhaBAD-lysY): This construct is referred to as pLemo in Wagner, et al. (2008) Proc. Natl. Sci. USA 105:14371-14376. Tunable T7 expression is achieved by modulating the level of T7 lyosyme (LysY) via L-rhamnose induction of the rhaBAD promoter.

The Lemo System™ is owned by Xbrane Biosciences AB (Stockholm, Sweden) and has been exclusively licensed to New England Biolabs, Inc. for the manufacture and distribution of Lemo21(DE3) Component E. coli.

The purchase of this product conveys to the non-commercial purchaser the non-transferable right to use the purchased amount of the product and components of the product for research use only conducted by the researcher. For commercial purchasers using this product for research, the purchase of this product conveys to the purchaser the non-transferable right to use the purchased amount of the product and components of the product for a six (6) month evaluation period after the date of purchase for research use only. Prior to the six (6) month evaluation, NEB or Xbrane Biosciences AB may contact the commercial purchaser regarding its continued use of this product for either commerical research or commercial purposes which require a commercial license. For more information, contact New England Biolabs, Inc. at busdev@neb.com. Commercial research and/or commercial purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. If the Purchaser is unwilling to accept the limitations of the limited-use statement, NEB is willing to accept return of the products with a full refund. 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.

RESEARCH USE ASSURANCE STATEMENT

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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