One Shot™ BL21-AI™ Chemically Competent E. coli
One Shot&trade; BL21-AI&trade; Chemically Competent <i>E. coli</i>
Invitrogen™

One Shot™ BL21-AI™ Chemically Competent E. coli

One Shot BL21-AI Chemically Competent E. coli cells are designed for applications that require tight regulation and strong expression ofRead more
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Catalog number C607003
Price (USD)
498.00
Each
Add to cart
Price (USD)
498.00
Each
Add to cart

One Shot BL21-AI Chemically Competent E. coli cells are designed for applications that require tight regulation and strong expression of toxic proteins from any T7-promoter-based expression systems. A derivative of the E. coli B strain, BL21-AI does not contain the lon protease and is deliberately deficient in the outer membrane protease OmpT.

The lack of two key proteases reduces degradation of heterologous proteins expressed in the strain. Additionally, several other strain features make these cells especially well 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.

BL21-AI E. coli contain a chromosomal insertion of a cassette containing the T7 RNA polymerase (T7RNAP) into the araB locus of the araBAD operon, placing regulation of T7 RNAP under the control of the arabinose-inducible araBAD promoter. The araB gene is deleted in this strain. The presence of the tetA gene confers resistance to tetracycline and permits verification of strain identity using tetracycline. The yield of recombinant protein obtained from BL21-AI is generally similar to that obtained from BL21 (DE3). BL21-AI, due it’s qualities and genomic background, was used to elucidate fundamental steps in the adaptation process of the CRISPR/Cas system. One Shot BL21-AI Chemically Competent cells have a transformation efficiency of >1 x 108 cfu/μg plasmid DNA.

One Shot BL21-AI Chemically Competent E. coli offer:
• Transformation efficiency of >1 x 108 cfu/μg control plasmid DNA
• Ion and OmpT protease deficiency that reduces degradation of recombinant protein
• T7 RNA polymerase controlled by the tightly regulated arabinose-inducible araBAD promoter
• hsdS mutation that allows efficient transformation of unmethylated DNA
• Ideal for inducing expression of toxic protein from any T7 promoter-based plasmid

Easy-to-use One Shot format
BL21-AI 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 (rB–, mB–) gal dcm araB::T7RNAP-tetA

Find the strain and format that fits your needs
We offer several E. coli strains for protein expression.
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.

Notes
• To induce expression from the araBAD promoter, use L-arabinose. To repress basal expression from the araBAD promoter, use glucose. Even if glucose is absent, basal expression from is generally low.
• BL21-AI strain genome sequences available in the NCBI GenBank database under accession number CP047231 (Bhawsinghka et al. 2020).

For Research Use Only. Not for use in diagnostic procedures.
Specifications
Product TypeChemically Competent Cells
Contains F' EpisomeNo
Improves Plasmid QualityNo
Cloning Methylated DNANo
Transformation Efficiency LevelMedium Efficiency (1 x 108 to 1 x 109 cfu/μg)
Antibiotic Resistance BacterialYes (Tetracycline)
Blue/White ScreeningNo
High-throughput CompatibilityLow
Preparing Unmethylated DNAYes (dcm)
Reduces RecombinationNo
Shipping ConditionDry Ice
T1 Phage - Resistant (tonA)No
SpeciesE. coli (B)
PromoterT7
FormatTube
Product LineOne Shot™
Quantity21 x 50 μL
Toxic ProteinsYes (araB)
Improves RNA StabilityNo
Improves Protein StabilityYes (lon, ompT)
Unit SizeEach
Contents & Storage
• BL21-AI One Shot Chemically Competent E. coli (21 x 50 μL); store at –80°C
• 20% L-arabinose in sterile water; store at –80°C
• pUC19 Control DNA (50 μL); store at –80°C
• S.O.C. Medium (6 mL); store at 4°C or room temperature

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 One Shot™ BL21-AI™ Chemically Competent E. coli FAQs