New England Biolabs, C2528J, Lemo21(DE3) Competent E. coli

Chemically competent Related Categories T7 Expression,, E. coli Protein Expression Strains,, E. coli, Expression Strains, Applications T7 Expression,, Toxic Protein Expression,, Membrane Protein Expression, Specification 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 FAQ Q: What are the solutions/recipes (C2528)? A: SOB: 2% Vegetable peptone (or Tryptone) 0.5% Yeast extract 10 mM NaCl 2.5 mM KCl 10 mM MgCl2 10 mM MgSO40.17 M NaCl LB agar: 1% Tryptone 0.5% Yeast Extract 1.5% Agar Q: What are the strain properties (C2528)? A: The properties of this strain that contribute to its usefulness as a protein expression strain are described below. The genotypes underlying these properties appear in parentheses. T7 RNA Polymerase: (T7 gene1) is encoded by the lambda DE3 prophage present within the chromosome. T7 RNA polymerase is expressed from the lacUV5 promoter, which is less sensitive to catabolite repression than the wt lac promoter. Thus DE3 strains may exhibit uninduced target protein expression. This is remedied by expression of T7 lysozyme (LysY). lysY: gene encoding the K128Y variant of phage T7 lysozyme. LysY lacks amidase activity yet retains the ability to inhibit T7 RNA polymerase. Protease Deficient ([lon] ompT): E. coli B strains are “naturally” deficient in the lon protease which in K-12 strains serves to degrade misfolded proteins and to prevent some cell cycle-specific proteins from accumulating. The OmpT protease resides at the surface of wild type E. coli in both K-12 and B strains, presumably helping the cells to derive amino acids from their external environment. Cells deficient in both these proteases are much more amenable to the production of proteins from cloned genes. T1 Phage Resistant (fhuA2): T1, an extremely virulent phage requires the E. coli ferric hydroxamate uptake receptor for infectivity. Deletion of this gene confers resistance to this type of phage, but does not significantly affect the transformation or growth characteristics of the cell. Q: Why are there no colonies or no growth in liquid culture (C2529)? A: This outcome is often due to basal expression of a target gene product, which is detrimental to cell viability. Basal expression of target protein in NiCo21(DE3) is identical to basal expression in BL21(DE3). Some vector systems (in particular those utilizing a T7 promoter) allow expression without inducer. If tightly regulated T7 expression is required, use a strain expressing lysY: T7 Express lysY (NEB #C3010) lysY produces a mutant T7 lysozyme which binds T7 RNA polymerase, reducing basal expression of the target protein. Upon induction, newly made T7 RNA polymerase titrates out the lysozyme and results in expression of the target protein. T7 Express lysY/Iq (NEB #C3013) lysY expression as well as lacI over-expression to repress basal expression of the T7 RNA polymerase. Lemo21(DE3) (NEB #C2528) BL21(DE3) containing the Lemo System™. LysY expression is modulated by L-rhamnose, making T7 protein expression tightly regulated and tunable. Q: Why is induced protein insoluble (C2528)? A: Check for insolubility – this is important because uncontrolled T7 expression often leads to very high production of protein that can result in the target protein becoming insoluble. Solutions around this are: Induce at a lower temperature (20 to 30°C) Induce earlier in growth phase (OD600 = 0.3 or 0.4) Test expression using a full range of L-rhamnose concentrations: 0 to 2,000 µM (see figure on the main product page). Q: Why is there no protein visible on gel or no activity (C2528)? A: Check for toxicity - no protein may mean the cells have eliminated or deleted elements in the expression plasmid. Culture cells for protein induction. Just before induction, plate a sample on duplicate plates with and without antibiotic selection. If toxicity is an issue, there will be a significant difference between the number of colonies on the plates. Fewer colonies will be seen on plates containing antibiotic (indicating that the plasmid has been lost) compared to plates without antibiotic. Check clone integrity by restriction enzyme analysis and/or sequencing of the ORF. If toxicity is the problem, add 500 µM L-rhamnose into the selection plates and into the starter culture media. Q: Can I store competent cells at -20°C instead of -80°C? A: Competent cells should be stored at -80°C. Storage at -20°C will result in a significant decrease in transformation efficiency (TE). When tested on NEB 5-alpha Competent E.coli (NEB #C2987H), cells lost 94.5% of TE after only 24 hours of storage at -20°C. Cells lost 98.9% of TE after 2 days, and 99.6% of TE after one week of storage at -20°C. Q: Which kind of transformation tubes should be used? A: Compared to 2.0 ml tube provided with NEB single-use format competent cells, the 1.5 ml Eppendorf tube we tested worked just fine. Q: What volume of DNA can be added into competent cells? A: The volume of DNA to be added into competent cells does affect transformation efficiency. 1-5 µl of DNA (plasmid or ligation product) is recommended for 50 µl of competent cells. In 50 µl of competent cells, transformation efficiency drops to 52% when the DNA volume is increased to 10 µl (from 2 µl). Transformation efficiency drops to 18% when the DNA volume is increased to 20 µl (from 2 µl). Transformation efficiency drops to 5.2% when the DNA volume is increased to 50 µl (from 2 µl). Q: What is the shelf life for this strain (NEB #C2528H)? A: The expiration date is one year from the assay date provided with the product. Q: Are NEB's competent cells compatible with the “Mix & Go" protocol? A: There is a “ Mix and Go" protocol that provides a quick way to transform your cells by simply adding plasmid to cells and plating. No heat shock step is required. NEB has tested our competent cells in this protocol against another company's “Mix & Go” product. We have observed both will produce similar numbers of colonies; however, the NEB colonies are larger in size using the same incubation period. Q: Is T7 expression subject to catabolite repression in BL21(DE3) and derivative strains? A: The lambda DE3 prophage carries the T7 RNA polymerase gene (T7gene1). Expression of the T7 RNA Polymerase is controlled by the lacUV5 promoter. Therefore, glucose addition will result in catabolite repression and basal protein expression from T7 promoter vectors will be better controlled when glucose is present in the media. When glycerol is the primary carbon source, there should be no effect on basal T7 expression.