New England Biolabs, C3030J, SHuffle® T7 Express lysY Competent E. coli

Having trouble opening your tubes? Related Categories SHuffle Expression,, E. coli Protein Expression Strains,, E. coli, Expression Strains, Applications T7 Expression,, Expression of Difficult Proteins,, Disulfide-bonded 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 Tetracycline 15 µg/ml Shipping Notes Ships on dry ice FAQ Q: What are the strain properties (C3030)? 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. Disulfide bond formation in the cytoplasm: Normally reductases in the E. coli cytoplasm keep cysteines in their reduced form, thereby reducing any disulfide bond that may form in this compartment. SHuffle has deletions of the genes for glutaredoxin reductase and thioredoxin reductase (Δgor ΔtrxB), which allows disulfide bonds to form in the cytoplasm. This combination of mutations is normally lethal, but the lethality is suppressed by a mutation in the peroxiredoxin enzyme (ahpC*). In addition, SHuffle expresses a version of the periplasmic disulfide bond isomerase DsbC which lacks its signal sequence, retaining it in the cytoplasm. This enzyme has been shown to act on proteins with multiple disulfide bonds, to correct mis-oxidized bonds and promote proper folding. The gene for the cytoplasmic DsbC is present on the chromosome. 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 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. Q: What applications are SHuffle® strains useful for? A: SHuffle strains are ideal for the expression of proteins that require disulfide bonds for their folding. The DsbC isomerase present in the chromosome of SHuffle strains has also been shown to be an effective chaperone (Chen et al. (1999) JBC, 274, 19601-19605) and might assist in the folding of target proteins, independent of disulfide bond formation. (de Marco, A. (2009) Microbial Cell Factories, 8, 26) Q: Does my protein have disulfide bonds? A: All known eukaryotic and prokaryotic cytoplasm contain reductases which participate in reducing disulfide bonds. Thus, proteins are oxidized to form disulfide bonds only in certain extra-cytoplasmic compartments such as the periplasmic space in gram negative prokaryotes or endoplasmic reticulum in eukaryotes. Exceptions to this may occur in certain thermophilic archaea such as Crenarchaea and few thermophilic bacteria (e.g. Aquifex and Thermotoga). There are websites such as Phobius or SignalP that can be used to predict whether a protein is in the oxidizing periplasm or endoplasmic reticulum. One can get an idea of the importance of cysteines in a protein of interest by analyzing how conserved the cysteine residues are in close homologs. Q: Which SHuffle® strain should I use? A: There are two versions of SHuffle® based on two different E. coli strains. SHuffle® T7 (NEB #C3026) is based on E. coli K12 while SHuffle® Express (NEB# C3028), SHuffle® T7 Express (NEB# C3029) and SHuffle® Express T7 LysY (NEB# C3030) are all based on E. coli B. We have observed varying levels of folding and expression within the two different SHuffle® cell lines (K12 vs B), depending on the protein. Currently we can not predict which SHuffle® cell line will be more successful in folding a given disulfide-bonded protein. We therefore recommend comparing the expression of your protein of interest in both SHuffle® strains (NEB #C3026 and #C3029) as the initial experiment. Once the optimum SHuffle® strain type is decided, optimization of expression conditions (e.g. temperature, inducer strength and timing, etc.) is recommend. All SHuffle strains can be used for non-T7 promoters (e.g. lac and ara promoter expression vectors). For T7 promoter expression vectors, we recommend either SHuffle T7 Express (NEB #C3029) or SHuffle T7 (NEB #C3026). For toxic T7 promoter expression, we recommend SHuffle® T7 Express lysY (NEB #C3030). Q: Is there anything I can do to increase protein yield when using SHuffle strains? A: Try varying the induction strength, duration and temperature to find optimum expression conditions. Lowering the expressing strength may improve yield. Q: What are the growth characteristics of the SHuffle strains? A: SHuffle strains reach similar final ODs as their wild type parental strains. However, we have observed a prolonged lag phase in SHuffle strains compared to wild type cells. Q: How do SHuffle® strains aid in cytoplasmic disulfide bond formation? A: SHuffle is a mutant E.coli strain lacking the two reductases (trxB and gor) with an additional suppressor mutation (ahpC) which restores viability, allowing the formation of stable disulfide bonds in the cytoplasm. Under these conditions thioredoxins are in their oxidized state, converting them from reductases to oxidases. Proteins that require disulfide bonds for their folding thus can be oxidized and form stable disulfide bonds within the cytoplasm. Additionally, SHuffle strains express the disulfide bond isomerase DsbC within the cytoplasm. This feature greatly enhances the fidelity of disulfide bond formation in the cytoplasm, and proteins with multiple disulfide bonds are correctly oxidized to significantly higher yields. 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: How should I express my protein of interest in SHuffle? A: For initial conditions we recommend using rich media at 30°C. Otherwise, overnight at 16°C is possible. At 30°C or 16°C, inoculate 1% overnight culture and grow cells at 30°C for 3 hours until OD600 ~ 0.8 and then induce expression of protein for at least 5 hours at 30°C or overnight at 16°C. If using 37°C, inoculate 1% overnight culture and grow cells for 2 hours at 37°C until OD600~0.8 and then induce expression of protein for at least 6 hours at 37°C. Q: Does Streptomycin and/or Spectinomycin need to be added when growing SHuffle strains? A: No, resistance to these antibiotics is due to gene(s) stably integrated into the chromosome. Expression plasmids with Streptomycin or Spectinomycin resistance markers should be avoided. Q: Can I conduct M13 phage display experiments in SHuffle strains? A: No. SHuffle Express cells (C3028, C3029, C3030) do not harbor the F' episome and therefore can not be infected by M13. On the other hand, even though SHuffle T7 cells (C3026) carry the F', they are not susceptible to M13 infection. This may be due to the oxidative cytoplasm interfering with phage assembly Q: Can I conduct blue/white screening using alpha complementation of lacZ in SHuffle strains? A: No, SHuffle strains do not have the appropriate M15 deletion in the lacZ gene for alpha-complementation. SHuffle (E. coli K12, C3026) and SHuffle express (E. coli B, C3028) cells are lacZ+, while SHuffle T7 express (E. coli B, C3029 and C3030) cells are lacZ-. Q: Which SHuffle strains are resistant to chloramphenicol? Is chloramphenicol required for maintenance of the mini F plasmid? A: Only SHuffle® T7 Express lysY Competent E. coli (NEB #C3030) are resistant to Cam. The Cam resistance is due to the miniF which carries the lysY gene. SHuffle® T7 Express Competent E. coli (NEB #C3029) is sensitive to Cam. It is not necessary to add Cam to propagate the miniF. We do not recommend adding Cam to cultures of SHuffle® T7 lysY Competent E. coli (NEB #C3027) or SHuffle® T7 Express lysY Competent E. coli (NEB #C3030). There is one copy of the Cam resistance gene on the miniF therefore standard concentrations of Cam (33 micrograms per ml) cannot be used. If chloramphenicol is added, use only 10ug/mL Q: Do SHuffle cells grow in minimal media? A: SHuffle express (E. coli B) cells (C3028, C3029, C3030) will grow in minimal media, while SHuffle (E. coli K12) is an auxotroph and will require the addition of Isoleucine and Leucine (50 ug/ml) for growth in minimal media. 100X stock solutions of Ile/Leu can be made by dissolving 5 mg/ml of the amino acids in water and filter sterilizing the solution. Q: Are SHuffle strains temperature sensitive? A: SHuffle express cells are sensitive to elevated temperatures and do no grow at 42º C. But at temperatures 37º C and below, both SHuffle express (E. coli B) and SHuffle (E. coli K12) cells are viable. We therefore suggest growing SHuffle cells at non-stressed temperatures of 30º C and induce expression of your protein at even lower temperatures Q: Is the genome of SHuffle strains available? A: Yes. You can read the paper that describes the complete annotated genome of both the K-12 version of SHuffle (C3026) and the B version (C3029) in Genome Announcements (2016) Mar 31;4(2). The annotated complete genome sequences are deposited at EMBL, EBI, under the following assembly accession numbers: NEB T7 express (C2566), CP014268; SHuffle-B (C3029), CP014269; DHB4, CP014270, and its cognate F′ episome, CP014271; and SHuffle K-12 (C3026), CP014272, and its cognate F′ episome, CP014273. 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. Q: Can one use SHuffle cells to secrete my protein of interest? A: Although SHuffle cells have an intact secretion apparatus, periplasmic production of proteins using SHuffle cells is not recommended. Proteins that require disulfide bonds for their folding can be oxidized in the cytoplasm, prior to its secretion, blocking the secretion of the protein.