One Shot™ BL21(DE3) Chemically Competent E. coli
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One Shot&trade; BL21(DE3) Chemically Competent <i>E. coli</i>
Invitrogen™

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

One Shot BL21(DE3) Chemically Competent E. coli cells are ideal for use with bacteriophage T7 promoter-based expression systems such as深入閱讀
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產品號碼Quantity
C60000321 x 50 μL/tube
產品號碼 C600003
價格 (TWD)
-
Quantity:
21 x 50 μL/tube
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One Shot BL21(DE3) Chemically Competent E. coli cells are ideal for use with bacteriophage T7 promoter-based expression systems such as pRSET and pET. This strain had been engineered to carry the λDE3 lysogen that contains T7 RNA polymerase gene under control of the lacUV5 promoter. Recombinant proteins that are nontoxic to E. coli are generally expressed at higher levels in BL21(DE3) cells compared to other strains with tighter expression control.

The BL21(DE3) E. coli strain is one of the most popular host strains to produce recombinant proteins and adapted in research or industrial applications. It 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 make 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. One Shot BL21(DE3) Chemically Competent E. coli cells can achieve transformation efficiency of >1 x 108 cfu/μg of control DNA.

Notes
• The basal expression levels of heterologous genes are significantly higher in BL21(DE3), thus strains such as BL21 AI are recommended for toxic protein expression.
• IPTG is required to induce expression of the T7 RNA polymerase cascade system that in turn transcribe T7 promoter regulated target gene.

One Shot BL21(DE3) Chemically Competent E. coli offer:
• Transformation efficiency of >1 x 108 cfu/μg control plasmid DNA 
• Ion and OmpT protease deficiency reduces degradation of recombinant protein 
• IPTG-inducible lacUV5 promoter controls expression of the T7 RNA polymerase 
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(DE3) 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 (DE3)

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.
規格
Antibiotic Resistance BacterialNo
Blue/White ScreeningNo
Cloning Methylated DNANo
Contains F' EpisomeNo
High-throughput CompatibilityLow
Improves Plasmid QualityNo
Improves Protein StabilityYes (lon, ompT)
Improves RNA StabilityNo
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
內容物與存放
• One Shot BL21(DE3) Chemically Competent E.coli (21 x 50 μL); store at –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

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

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常見問答集 (常見問題)

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.

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引用資料與參考文獻
Abstract
The interaction between HIV-1 Gag and human lysyl-tRNA synthetase during viral assembly.
Authors:Javanbakht H, Halwani R, Cen S, Saadatmand J, Musier-Forsyth K, Gottlinger H, Kleiman L,
Journal:J Biol Chem
PubMed ID:12756246
'Human lysyl-tRNA synthetase (LysRS) is a tRNA-binding protein that is selectively packaged into HIV-1 along with its cognate tRNALys isoacceptors. Evidence exists that Gag alone is sufficient for the incorporation of LysRS into virions. Herein, using both in vitro and in vivo methods, we begin to map regions in Gag ... More
The anti-trp RNA-binding attenuation protein (Anti-TRAP), AT, recognizes the tryptophan-activated RNA binding domain of the TRAP regulatory protein.
Authors: Valbuzzi Angela; Gollnick Paul; Babitzke Paul; Yanofsky Charles;
Journal:J Biol Chem
PubMed ID:11786553
'In Bacillus subtilis, the trp RNA-binding attenuation protein (TRAP) regulates expression of genes involved in tryptophan metabolism in response to the accumulation of l-tryptophan. Tryptophan-activated TRAP negatively regulates expression by binding to specific mRNA sequences and either promoting transcription termination or blocking translation initiation. Conversely, the accumulation of uncharged tRNA(Trp) ... More
The ISG15 isopeptidase UBP43 is regulated by proteolysis via the SCFSkp2 ubiquitin ligase.
Authors:Tokarz S, Berset C, La Rue J, Friedman K, Nakayama K, Nakayama K, Zhang DE, Lanker S,
Journal:J Biol Chem
PubMed ID:15342634
'The Skp2 oncoprotein belongs to the family of F-box proteins that function as substrate recognition factors for SCF (Skp1, cullin, F-box protein) E3 ubiquitin-ligase complexes. Binding of the substrate to the SCFSkp2 complex catalyzes the conjugation of ubiquitin molecules to the bound substrate, resulting in multi-ubiquitination and rapid degradation by ... More
A proteolytic transmembrane signaling pathway and resistance to beta-lactams in staphylococci.
Authors:Zhang HZ, Hackbarth CJ, Chansky KM, Chambers HF.
Journal:Science
PubMed ID:11239156
beta-Lactamase and penicillin-binding protein 2a mediate staphylococcal resistance to beta-lactam antibiotics, which are otherwise highly clinically effective. Production of these inducible proteins is regulated by a signal-transducing integral membrane protein and a transcriptional repressor. The signal transducer is a fusion protein with penicillin-binding and zinc metalloprotease domains. The signal for ... More
The solution structure and interactions of CheW from Thermotoga maritima.
Authors: Griswold Ian J; Zhou Hongjun; Matison Mikenzie; Swanson Ronald V; McIntosh Lawrence P; Simon Melvin I; Dahlquist Frederick W;
Journal:Nat Struct Biol
PubMed ID:11799399
Using protein from the hyperthermophile Thermotoga maritima, we have determined the solution structure of CheW, an essential component in the formation of the bacterial chemotaxis signaling complex. The overall fold is similar to the regulatory domain of the chemotaxis kinase CheA. In addition, interactions of CheW with CheA were monitored ... More
12 total citations

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