BL21 Star™ (DE3)pLysS One Shot™ Chemically Competent E. coli

BL21 Star™ (DE3)pLysS One Shot™ Chemically Competent <i>E. coli</i>

Invitrogen™

BL21 Star™ (DE3)pLysS One Shot™ Chemically Competent E. coli

One Shot BL21 Star (DE3)pLysS Chemically Competent E. coli are cells designed for applications that require high-level expression of non-toxicRead more

Catalog Number	Quantity
C602003	21 x 50 μL/tube

Catalog number C602003

Price (JPY)

91,200

Quantity:

21 x 50 μL/tube

One Shot BL21 Star (DE3)pLysS Chemically Competent E. coli are cells designed for applications that require high-level expression of non-toxic recombinant proteins from low or high copy number vectors, such as pRSET and pET. The BL21 (DE3) E. coli strain is one of the most popular host strains to produce recombinant proteins and is adapted for research and industrial applications (e.g., first biopharmaceutical recombinant human insulin).

BL21(DE3) is a derivative of the E. coli B strain that does not contain the lon protease and is also deficient in the outer membrane protease OmpT. The lack of two key proteases reduces degradation of heterologous proteins expressed in the strains. Additionally, several other strain features made them especially suited for protein production, namely fast growth in minimal medium, a lower acetate production when grown in high glucose media, and the ability to reach high cell density.

This strain was engineered to carry the λDE3 lysogen that contains T7 RNA polymerase gene under control of the lacUV5 promoter. IPTG is required to induce expression of the T7 RNA polymerase cascade system that in turn transcribe T7 promoter regulated target gene. The pLysS CamR plasmid carried by the BL21 Star (DE3) pLysS strain expresses T7 lysozyme, a T7 RNA polymerase inhibitor that prevents leaky expression in uninduced cells. The p15a origin of pLysS makes this plasmid compatible with pUC or pBR322-derived plasmids.This strain offers enhanced mRNA stability due to a mutation in the RNaseE gene (rne131) that reduces levels of endogenous RNases and mRNA degradation, thereby increasing the stability of mRNA transcripts and increasing protein yield. The BL21 Star (DE3) pLys strain has lower basal expression of heterologous genes than BL21 Star (DE3) strain. Other trains such as BL21-AI are recommended for toxic protein expression. One Shot BL21 Star (DE3) pLysS Chemically Competent cells have a transformation efficiency of >1 x 10⁸ cfu/μg plasmid DNA.

One Shot BL21 Star (DE3)pLysS Chemically Competent E. coli offer:
• Transformation efficiency of >1 x 10⁸ cfu/μg plasmid DNA
• Ion and OmpT protease deficiency reduces degradation of recombinant protein
• IPTG-inducible lacUV5 promoter controls expression of the T7 RNA polymerase
• rne131 mutation reduces levels of endogenous RNases and mRNA degradation
• pLysS (CamR)plasmid for reduced basal expression in uninduced cells
• hsdS mutation allows efficient transformation of unmethylated DNA
• Fast growth in minimal medium and ability to reach high cell density

Easy-to-use One Shot format
BL21 Star (DE3) pLysS Chemically Competent E. coli cells are supplied in the convenient, single-reaction One Shot format. The single-tube, single-use format allows all steps of the transformation protocol, up to plating, to take place in the same tube, thereby helping save time and prevent contamination.

Genotype
F^-ompT hsdSB (r_B^-, m_B^-) gal dcm rne131 (DE3) pLysS (CamR)

Find the strain and format that you need
We offer other E. coli strains for protein expression.
For expression of toxic proteins, consider BL21-AI One Shot Chemically Competent E. coli.
BL21 Star (DE3) and other strains are available in MultiShot formats for high throughput applications.
Explore bacterial growth media formats.
We offer a variety of systems for the expression of recombinant proteins in E. coli, the Champion pET expression system provides the highest level of protein production available in any expression system.

For Research Use Only. Not for use in diagnostic procedures.

Specifications

Antibiotic Resistance BacterialYes (Chloramphenicol)

Blue/White ScreeningNo

Cloning Methylated DNANo

Contains F' EpisomeNo

High-throughput CompatibilityLow

Improves Plasmid QualityNo

Improves Protein StabilityYes (lon, ompT)

Improves RNA StabilityYes (rne131, pLysS)

Preparing Unmethylated DNAYes (dcm)

Product LineOne Shot™

Product TypeChemically Competent Cells

Quantity21 x 50 μL/tube

Reduces RecombinationNo

Shipping ConditionDry Ice

T1 Phage - Resistant (tonA)No

Toxic ProteinsNo

Transformation Efficiency LevelMedium Efficiency (1 x 10⁸ to 1 x 10⁹ cfu/μg)

FormatTube

PromoterT7

SpeciesE. coli (B)

Unit SizeEach

Contents & Storage

• BL21 Star (DE3)pLysS Chemically Competent E. coli (21 x 50 μL); storeat –80°C
• pUC19 DNA (1 x 50 μL); store at –20°C
• S.O.C. Medium (6 mL); store at 4°C or room temperature

Ready-to-use Bacterial Growth Media

See how these mediums can help with critical aspects of cloning!

Gibco LB Broth ›

Invitrogen S.O.C. Medium ›

Invitrogen One Shot LB Agar* ›

*Only available in North America and selected European countries

Frequently asked questions (FAQs)

My gene of interest is toxic to bacterial cells. Are there any precautions you can suggest?

Several precautions may be taken to prevent problems resulting from basal level expression of a toxic gene of interest. These methods all assume that the T7-based or Champion-based expression plasmid has been correctly designed and created.

- Propagate and maintain your expression plasmid in a strain that does not contain T7 RNA polymerase (i.e., DH5α).
- If using BL21 (DE3) cells, try growing cells at room temperature rather than 37 degrees C for 24-48 hr.
- Perform a fresh transformation using a tightly regulated E. coli strain, such as BL21-AI cells.
- After following the transformation protocol, plate the transformation reaction on LB plates containing 100 µg/mL ampicillin and 0.1% glucose. The presence of glucose represses basal expression of T7 RNA polymerase.
- Following transformation of BL21-AI cells, pick 3 or 4 transformants and inoculate directly into fresh LB medium containing 100 µg/mL ampicillin or 50 µg/mL carbenicillin (and 0.1% glucose, if desired). When the culture reaches an OD600 of 0.4, induce expression of the recombinant protein by adding L-arabinose to a final concentration of 0.2%.
- When performing expression experiments, supplement the growth medium with 0.1% glucose in addition to 0.2% arabinose.
- Try a regulated bacterial expression system such as our pBAD system.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I'm trying to express my protein using a bacterial expression system. How do I know if I'm seeing degradation of my protein or if what I’m seeing is codon usage bias?

Typically, if you see 1-2 dominant bands, translation stopped prematurely due to codon usage bias. With degradation, you usually see a ladder of bands. With degradation, you can try using a protease inhibitor and add it to the lysis buffer to help prevent degradation. If degradation is the issue, a time point experiment can be done to determine the best time to harvest the cells.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I'm trying to express my protein using a bacterial expression system and am getting inclusion bodies. What should I do?

If you are having a solubility issue, try to decrease the temperature or decrease the amount of IPTG used for induction. You can also try a different, more stringent cell strain for expression. Adding 1% glucose to the bacterial culture medium during expression can also help.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

I'm getting low protein yield from my bacterial expression system. What can I do to improve this?

- Inoculate from fresh bacterial cultures, since higher protein yields are generally obtained from a fresh bacterial colony.

- Check the codon usage in the recombinant protein sequence for infrequently used codons. Replacing the rare codons with more commonly used codons can significantly increase expression levels. For example, the arginine codons AGG and AGA are used infrequently by E. coli, so the level of tRNAs for these codons is low.

- Add protease inhibitors, such as PMSF, to buffers during protein purification. Use freshly made PMSF, since PMSF loses effectiveness within 30 min of dilution into an aqueous solution.

- If you are using ampicillin for selection in your expression experiments, you may be experiencing plasmid instability due to the absence of selective conditions. This occurs as the ampicillin is destroyed by β-lactamase or hydrolyzed under the acidic media conditions generated by bacterial metabolism. You may want to substitute carbenicillin for ampicillin in your transformation and expression experiments.

- The recombinant protein may be toxic to bacterial cells. Try a tighter regulation system for competent cell expression such as BL21-AI. You may also consider trying a different expression system such as the pBAD system.

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

My cells are growing very slowly, and I'm not getting any protein expression from my baterial expression system. What can I do to fix this?

This typically occurs when your gene of interest is toxic. Try using a tighter regulation system, such as BL21 (DE3) (pLysS) or BL21 (DE3) (pLysE), or BL21(AI).

Find additional tips, troubleshooting help, and resources within our Protein Expression Support Center.

View all BL21 Star™ (DE3)pLysS One Shot™ Chemically Competent E. coli FAQs