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Merck
CN

B3310

Sigma-Aldrich

e coli protein expression

for protein expression and DNA plasmid production

Synonym(s):

BL21 Competent Cells, BL21 (DE3)pLysS-T1R Competent Cells

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About This Item

MDL number:
MFCD01634471
UNSPSC Code:
12352200

Key Documents

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Molecular Biology

form

suspension

shipped in

dry ice

storage temp.

−70°C

General description

BL21(DE3)pLysS-T1R are competent E. coli that are suitable for high level induction and expression genes regulated by expression vectors with T7 promoter. The cells have transformation efficiency of ≥5x106 cfu/μg when transformed with non-saturating amounts of pUC19 plasmid DNA.
Sigma′s BL21-T1R competent E. coli cells are grown and made chemically competent using an optimized procedure specific to the strain, followed by strain verification and efficiency testing. The cells are provided in frozen 50 μl aliquots for convenience. Each aliquot can be used for a single transformation.

Application

Suitable for induction and expression of genes directed by the expression systems with T7 promoter

Biochem/physiol Actions

BL21(DE3)pLysS-T1R does not express ion proteases and outer membrane protease, ompT. This prevents the degradation of heterologous proteins expressed by T7 expression vector systems. This strain also contains tonA genotype that confers resistance to lytic bacteriophages such as T1 and T5. The strain is lysogenic for lambda prophage and contains an inducible T7 RNA polymerase regulated by lacUV5 promoter. T7 lysozyme produced by the pLysS plasmid inhibits T7 polymerase that improves transcriptional control.

Features and Benefits

  • Ensures induction and expression of genes from any T7-promoter based expression system
  • Contains the genotype tonA that protects the clonal stocks from lytic bacteriophages
  • Contains T7 RNA polymerase that is inducible by addition of IPTG to the culture
  • Renders resistance to chloramphenicol; useful for selection process
  • Allows compatibility with plasmids containing ColE1 or pMB1 origin
  • Guaranteed high transformation efficiency
  • Convenient 50 μL aliquots

Other Notes

  • BL21(DE3)pLysS-T1R chemically competent cells, 10 X 50 μL (B3185)
  • pUC 19 control DNA (10 ng/μL), 10 μL (D2567)

Storage Class Code

10 - Combustible liquids

Flash Point(F)

Not applicable

Flash Point(C)

Not applicable

Regulatory Information

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Certificates of Analysis (COA)

Lot/Batch Number
SLBL1311V
SLBG8829
SLBB6935
051M0968
090M0801

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  1. Which document(s) contains shelf-life or expiration date information for a given product?

    If available for a given product, the recommended re-test date or the expiration date can be found on the Certificate of Analysis.

  2. How do I get lot-specific information or a Certificate of Analysis?

    The lot specific COA document can be found by entering the lot number above under the "Documents" section.

  3. How do I find price and availability?

    There are several ways to find pricing and availability for our products. Once you log onto our website, you will find the price and availability displayed on the product detail page. You can contact any of our Customer Sales and Service offices to receive a quote.  USA customers:  1-800-325-3010 or view local office numbers.

  4. What is the Department of Transportation shipping information for this product?

    Transportation information can be found in Section 14 of the product's (M)SDS.To access the shipping information for this material, use the link on the product detail page for the product. 

  5. Are home-made competent cells as efficient at transformation as purchased cells?

    They can be, depending on the technique used, the expected transformation efficiency and how the cells are handled.  For special applications, competent cells prepared in-house using standard methods may not provide the efficiency you need.

  6. Are all competent cells suitable for both plasmid production and protein expression?

    No.  Most strains of competent cells are suitable for producing plasmid DNA that will be used to transfect insect or mammalian cells for expression of the protein.  The BL21 competent cell strains have special traits enabling protein expression in bacteria.

  7. What are the differences among the BL21 strains?

    The BL21-T1R cells are suitable for high level production of heterologous proteins directed by various expression vector systems (promoters such as lac, trc, tac, ?PL and araD).  The BL21 (DE3) - T1R cells are suitable for high level induction and expression of genes from any T7 promoter-based expression vector. DE(3) indicates that the strain is lysogenic for a lambda prophage containing an inducible T7 RNA polymerase under control of the lacUV5 promoter.The BL21 (DE3) pLysS - T1R cells are suitable for high level induction and expression of genes from any T7 promoter-based expression vector pLysS.  The cells express T7 lysozyme, a natural inhibitor of T7 polymerase, allowing for improved transcriptional control and reduction of "leaky" expression. The pLysS plasmid also renders the cell resistant to chloramphenicol (CmR) and contains the p15A origin. This allows pLysS to be compatible with plasmids containing the ColE1 or pMB1 origin.

  8. When using the competent cells for protein expression, what traits are required?

    Ideally, expression strains will minimally have the following genotype:EndA (DNA is not degraded)hsd (unmethylated DNA is not restricted)laclq (better control over the lac promoter)lon (lacks intracellular protease)ompT (lacks extracellular protease)pLysS (inhibits T7 RNA polymerase)tonA (resistant to T1 phage)trfA (replication of oriV plasmids)T7 RNA polymerase

  9. What are important considerations when performing bacterial transformation?

    Things to consider when planning bacterial transformation:DNA impuritiesSource of DNAAmount of DNA usedStorage and handling of the competent cells

  10. Can I store the competent cells in the -20C?

    No.  Competent cells should not be stored at -20C for any length of time.  The cells suffer a dramatic drop in transformation efficiency when stored higher than -80C.

  11. How should I thaw the competent cells?

    Competent cells should be thawed on ice.  The transformation efficiency is dependent on the proper handling of the cells (maintaining cold temperature until transformation).

  12. Can I re-freeze the vial if I do not use the entire aliquot of competent cells?

    Re-freezing competent cells will result in a decrease in their transformation efficiency.  If the cells are frozen first in a dry ice / ethanol bath before placement in the -80C freezer, the loss will be about two-fold.  If placed directly in the -80C freezer, the loss in transformation efficiency is about five to ten-fold.

  13. I’m working with BL21(DE3) pLysS and I’m getting a lot of background expression. Why?

    When using the BL21 (DE3) pLysS - T1R competent cells, selection should be performed for the introduced plasmid as well as the pLysS.  This should be done by placing chloramphenicol (Cm) in selective plates and growth media. If there is still alot of background when selecting with Cm, switch to a promoter system that has better control.

  14. Competent cells were left on ice overnight - can they still be used?

    Competent cells left on ice, or allowed to come to room temperature slowly will suffer a dramatic loss in transformation efficiency.  We recommend thawing a new aliquot of competent cells.

  15. Can I clone in BL21 cells?

    Yes, BL21 cells can be used for cloning.  If using a cloning method with high yield and low background, then transformation into BL21 cells can be performed directly.  If cloning by restriction digest, it is better to use a cloning strain first, before putting the plasmid into BL21.

  16. Can I express a recombinant protein that is toxic to the bacteria?

    For expression of toxic recombinant proteins, select the BL21 strain containing the pLysS plasmid. These cells express low levels of lysozyme that will bind to T7 polymerase, and inhibit transcription.  

  17. What DNA purity do I need for use in transformation?

    The DNA used should be high quality - free from phenol, proteins, detergents, and ethanol.  Dissolve the DNA in sterile water or 0.5x TE buffer (5 mM Tris-HCl, 0.5 mM EDTA).

  18. Can I grow more competent cells from the stock I purchased?

    Generally, no, because the future generations of cells lose their competency.

  19. My question is not addressed here, how can I contact Technical Service for assistance?

    Ask a Scientist here.

F W Studier et al.
Journal of molecular biology, 189(1), 113-130 (1986-05-05)
A gene expression system based on bacteriophage T7 RNA polymerase has been developed. T7 RNA polymerase is highly selective for its own promoters, which do not occur naturally in Escherichia coli. A relatively small amount of T7 RNA polymerase provided
Host specificity of DNA produced by Escherichia coli: bacterial mutations affecting the restriction and modification of DNA.
W B Wood
Journal of molecular biology, 16(1), 118-133 (1966-03-01)
T A Phillips et al.
Journal of bacteriology, 159(1), 283-287 (1984-07-01)
The product of the pleiotropic gene lon is a protein with protease activity and has been tentatively identified as protein H94.0 on the reference two-dimensional gel of Escherichia coli proteins. Purified Lon protease migrated with the prominent cellular protein H94.0

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