New England Biolabs, C3013I, T7 Express lysY/Iq Competent E. coli (High Efficiency)

High efficiency 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: Why are there no colonies or no growth in liquid culture (C3013)? A: Even though T7 expression is tightly regulated, there may be a low level of basal expression in the T7 Express host. If toxicity of the expressed protein is likely, transformation of the expression plasmid should be carried out in one of the following strains: » T7 Express Iq : over-expression of the LacI repressor reduces basal expression of the T7 RNA polymerase » T7 Express lysY : lysY produces mutant T7 lysozyme which binds to 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 Iq/lysY combines both above effects. Incubation at 30°C or room temperature may also alleviate toxicity issues. In addition, check antibiotic concentration (test with control plasmid). Q: Why is there no protein visible on gel or no activity (C3013)? 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. Q: Why is induced protein insoluble (C3013)? A: Check for insolubility - this is important because 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 lower temperatures (as low as 12 - 15°C overnight) » Reduce IPTG concentration to 0.01 - 0.1 mM » Induce for less time (as little as 15 minutes) » Induce earlier in growth (OD600 = 0.3 or 0.4) Q: What are the solutions/recipes (C3013)? A: SOB: 2% Vegetable peptone (or Tryptone) 0.5% Yeast Extract 10 mM NaCl 2.5 mMKCl 10 mM MgCl2 10 mM MgSO4 SOC: SOB + 20 mM Glucose LB agar: 1% Tryptone 0.5% Yeast Extract 0.17M NaCl 1.5% Agar 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: What is the difference between NEB #C3013H and NEB #C3013I? A: They are the same cells with the same efficiency but provided in different formats. C3013H is packaged with 20 single-use transformation tubes, each containing 50 μl of competent cells. Plasmid or ligation product can be added directly into the transformation tubes for convenience. C3013I is packaged with 6 tubes, each containing 200 μl of competent cells. The tubes should be thawed on ice and 50 μl of cells transferred into new tubes prior to transformation. Each tube contains enough cells for 4 transformations with the benefit of reducing the cost of each transformation. If you perform 3 or 4 transformations at a time, using C3013I is cost effective. Refreezing the competent cells after thawing is not recommended since it will significantly reduce transformation efficiencies. Q: What are the strain properties (C3013)? 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 (lacZ::T7 gene1): T7-Express has the T7 RNA polymerase gene inserted into the lac operon on the E. coli chromosome and is expressed under the control of the lac promoter. This configuration provides controlled induction of T7 RNA polymerase and, consequently, inducible control of transcription of genes downstream of the T7 promoter. This system provides potential advantages over strains such as BL21(DE3), that carry the T7 RNA polymerase on a lysogenic prophage. Although λDE3 is normally dormant in the host chromosome, the induction of the SOS cascade can occur as the result of expressing proteins that damage the E. coli chromosome, either directly or indirectly. This may lead to cell lysis. Lac Promoter Control (lacIq): The lac repressor blocks expression from lac, tac and trc promoters frequently carried by expression plasmids. If the level of lac repressor in E. coli cells is not sufficient to inhibit expression via these promoters during transformation or cell growth, even low levels of expression of toxic genes can reduce transformation efficiency and select against desired transformants. The extra molecules of lac repressor in lacIq strains help to minimize promoter activity until IPTG is added. T7 Lysozyme (lysY): This strain expresses T7 lysozyme variant K128Y which lacks amidase activity, yet retains the ability to inhibit T7 RNA polymerase. Basal expression of the target gene is minimized without inhibiting IPTG-induced expression. The lysY gene is carried on a single-copy miniF plasmid. 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. Recovery from DNA Damage (sulA11): E. coli cells can tolerate a substantial amount of chronic DNA damage as long as repair is allowed to proceed. This capacity is compromised if the cells are unable to divide following repair. In lon- cells, SulA, a cell division inhibitor, accumulates and causes cells to become hypersensitive to DNA damage. The sulA mutation introduced into the T7-Express strain allows cells to divide more normally in the absence of Lon protease. Endonuclease I Deficient (endA1): The periplasmic space of wild type E. coli cells contains the nonspecific endonuclease, EndA. Extreme care must be taken to avoid degradation of plasmids prepared from these cells. The endA mutation deletes this endonuclease and can significantly improve the quality of plasmid preparations. Restriction Deficient (Δ(mcrC-mrr)114::IS10): Wild type E. coli B strains carry a Type I restriction endonuclease which cleaves DNA with the site TGA(N8)TGCT. While E. coli DNA is protected from degradation by a cognate methyl-transferase, foreign DNA will be cut at these sites. The deletion described above eliminates both the methylase and the endonuclease. Methyl Restriction Deficient (Δ(mcrC-mrr)114::IS10 and R(mcr-73::miniTn10--TetS)2): E. coli has a system of enzymes encoded by mcrA, mcrBC and mrr which will cleave DNA with methylation patterns found in higher eukaryotes, as well as some plant and bacterial strains. All three Mcr enzymes and Mrr have been inactivated in T7 Express allowing the introduction of genomic DNA from such cells if desired. 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: What is the optimal heat shock time for this strain (NEB #C3013H and NEB #C3013I)? A: Heat shock at 42ºC for 10 seconds results in the highest transformation efficiency for T7 Express lysY/Iq competent E.coli (NEB #C3013H and NEB #C3013I). Expect approximately 50% loss in transformation efficiency when heat shocking for 80 seconds (see Figure on the main product page). Q: How long should I incubate cells on ice after DNA has been added (NEB #C3013H and NEB #C3013I)? A: Incubating DNA with T7 Express lysY/Iq competent cells on ice for 30 minutes is recommended. When tested with pUC19, an incubation time of just 2 minutes did not significantly lower the transformation efficiency (see Figure on the main product page). Q: Is T7 Express lysY/Iq (NEB #C3013H and NEB #C3013I) compatible with auto-induction procedures? A: T7 Express lysY/Iq is not compatible with auto-induction procedures. In auto-inducing media, lactose serves to induce expression of the T7 RNA polymerase upon conversion to allo-lactose by beta-galactosidase (the lacZ gene product). Therefore, auto-induction should be performed using strains with an intact lac operon (1). In T7 Express lysY/Iq strain the T7 RNA polymerase gene (T7 gene1) disrupts the lacZ gene causing inactivation of beta-galactosidase. Therefore T7 Express lysY/Iq strain is not recommended for auto-induction procedures. We recommend host strains BL21(DE3) (NEB #C2527H and NEB #C2527I) or NiCo21(DE3) (NEB #C2529H) for auto-induction procedures. (1) Studier, F. W. (2005) Protein Production by Auto-Induction in High-Density Shaking Cultures Protein Expr. Purif. 41, 207-34. 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 #C3013H and NEB #C3013I)? A: The expiration date is two years 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 antibiotic selection necessary to maintain the MiniF-lysY-lacIq plasmid? A: Although the MiniF plasmid carries the chloramphenicol resistance gene, addition of chloramphenicol is not necessary. If added, chloramphenicol should be at a final concentration of 10 μg/ml. Chloramphenicol levels greater than 15 μg/ml will impair growth. Q: Is T7 expression subject to catabolite repression in NEB T7 Express or SHuffle strains? A: The T7 RNA polymerase gene is cloned into the chromosomal lac operon. Since expression is controlled by the wt lac operon, glucose addition will result in catabolite repression. Thus, 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 no effect on the lac operon.