Datasheet for bl21(de3) competent e. coli (c2527; lot 30)
5 Minute Transformation Protocol
A shortened transformation protocol resulting in approximately 10% effi-ciency compared to the standard protocol may be suitable for applications Competent E. coli
where a reduced total number of transformants is acceptable.
Follow the Transformation Protocol with the following changes:1. Steps 3 and 5 are reduced to 2 minutes.
2. Omit outgrowth (step 7) completely for ampicillin-resistant plas- mids or reduce the outgrowth time for other selective media as Protocol for Protein Expression Using BL21(DE3)
1. Transform expression plasmid into BL21(DE3). Plate on antibiotic
6 x 0.2 ml/tube
selection plates and incubate overnight at 37°C.
Store at –80°C
2. Resuspend a single colony in 10 ml liquid culture with antibiotic. 3. Incubate at 37°C until OD reaches 0.4–0.8.
CAUTION: This product contains DMSO, a hazardous material. Review the
4. Induce with 4 or 40 µl of a 100 mM stock of IPTG (final concentra- MSDS before handling. tion of 40 or 400 µM) and induce for 3 to 5 hours at 37°C. Description: Chemically competent E. coli cells suitable for transformation
5. Check for expression either by Coomassie stained protein gel, and protein expression.
Western Blot or activity assay. Check expression in both the total cell extract (soluble + insoluble) and the soluble fraction only.
*If a fraction of the target protein is insoluble, repeat expression at a ❚ Transformation efficiency: 1–5 x 107 cfu/µg pUC19 DNA lower temperature (15 to 30°C) or test expression in Lemo21(DE3) (NEB #C2528).
❚ T7 Expression Strain 6. For large scale, inoculate 1 L of liquid medium (with antibiotic) with ❚ Deficient in proteases Lon and OmpT a freshly grown colony or 10 ml of freshly grown culture. Incubate ❚ Resistant to phage T1 (fhuA2) at 37°C until OD reaches 0.4–0.8. Add 40 or 400 µM IPTG and ex- press protein using optimal time/temperature determined in a small ❚ Free of animal products Reagents Supplied:
Troubleshooting T7 Protein Expression
6 x 0.2 ml/tube of chemically competent BL21(DE3) Competent E. coli cells No colonies or no growth in liquid culture: T7 expression in BL21(DE3)
(Store at –80°C)
is not tightly regulated and thus toxic proteins may affect cell viability, es-pecially if the expression vector does not encode lacI. For tightly regulated 25 ml of SOC Outgrowth Medium (Store at room temperature)
expression, use a strain expressing lysY: 0.025 ml of 50 pg/µl pUC19 Control DNA (Store at –20°C)
❚❚❚ T7 Express lysY (NEB #C3010): lysY produces mutant T7 lysozyme which binds to T7 RNA polymerase, reducing basal expression of the Quality Control Assays
target protein. Upon induction, newly made T7 RNA polymerase titrates Transformation Efficiency: 100 pg of pUC19 plasmid DNA was used to
out the lysozyme and results in expression of the target protein transform one tube of BL21(DE3) Competent E. coli following the protocol ❚❚❚ T7 Express lysY/Iq (NEB #C3013): lysY expression as well as lacI over- provided. 1–5 x 107 colonies formed/µg after an overnight incubation on expression to repress basal expression of the T7 RNA Polymerase.
LB-ampicillin plates at 37°C.
❚❚❚ Lemo21(DE3) (NEB #C2528): BL21(DE3) containing the Lemo Sys- Untransformed cells were also tested for resistance to phage ϕ80, a tem™. lysY expression is modulated by L-rhamnose, making T7 protein standard test for resistance to phage T1 and sensitivity to ampicillin, expression tightly regulated and tunable.
chloramphenicol, kanamycin, nitrofurantoin, spectinomycin, streptomycin and tetracycline. No protein visible on gel or no activity: Check for toxicity - no protein
may mean the cells have eliminated or deleted elements in the expression 1. Thaw a tube of BL21(DE3) Competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µl of cells into ❚❚ Culture cells for protein induction. Just before induction, plate a sample a transformation tube on ice.
on duplicate plates with and without antibiotic selection. If toxicity is 2. Add 1–5 µl containing 1 pg–100 ng of plasmid DNA to the cell mixture. an issue, there will be a significant difference between the number of Carefully flick the tube 4–5 times to mix cells and DNA. Do not vortex.
colonies on the plates. Fewer colonies will be seen on plates containing 3. Place the mixture on ice for 30 minutes. Do not mix. antibiotic (indicating that the plasmid has been lost) compared to plates without antibiotic.
4. Heat shock at exactly 42°C for exactly 10 seconds. Do not mix.
5. Place on ice for 5 minutes. Do not mix.
❚❚ Check clone integrity by restriction enzyme analysis and/or sequencing 6. Pipette 950 µl of room temperature SOC into the mixture. If toxicity is the problem, lysY expressing strains will provide clone 7. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.
8. Warm selection plates to 37°C.
(see other side) 9. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.
10. Spread 50–100 µl of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24–36 hours or at 25°C for 48 hours.
STORAGE AND HANDLING: Competent cells should be stored at –80°C. Storage at –20°C will result in a significant decrease in transformation efficiency. Cells lose efficiency whenever they are warmed above –80°C, even if they do not thaw.
CERTIFICATE OF ANALYSIS
Induced protein is insoluble: Check for insolubility - this is important
Transformation Protocol Variables
because T7 expression often leads to very high production of protein Thawing: Cells are best thawed on ice and DNA added as soon as the last bit
that can result in the target protein becoming insoluble. Solutions around of ice in the tube disappears. Cells can also be thawed by hand, but warming above 0°C will decrease the transformation efficiency.
❚ Induce at a lower temperature (as low as 15°C overnight) Incubation of DNA with Cells on Ice: For maximum transformation ef-
❚ Reduce IPTG concentration to 40 µM ficiency, cells and DNA should be incubated together on ice for 30 minutes. Expect a 2-fold loss in transformation efficiency for every 10 minutes you ❚ Induce earlier in growth phase (OD = 0.3 or 0.4) shorten this step.
❚ Test expression in Lemo21(DE3) – see example below: Heat Shock: Both the temperature and the timing of the heat shock step are
important and specific to the transformation volume and vessel. Using the transformation tube provided, 10 seconds at 42°C is optimal.
Titration of lysY Outgrowth: Outgrowth at 37°C for 1 hour is best for cell recovery and for
expression of antibiotic resistance. Expect a 2-fold loss in transformation ef-
ficiency for every 15 minutes you shorten this step. SOC gives 2-fold higher
transformation efficiency than LB medium; and incubation with shaking or
rotating the tube gives 2-fold higher transformation efficiency than incuba-
tion without shaking.
Plating: Selection plates can be used warm or cold, wet or dry without sig-
nificantly affecting the transformation efficiency. However, warm, dry plates
are easier to spread and allow for the most rapid colony formation.
0 250 500 750 1,000 2,000 Strain Properties
Western analysis of 6-His tagged Brugia malayi protein. A) B. malayi protein
The properties of this strain that contribute to its usefulness as a protein expressed at 20°C in BL21(DE3). B) Soluble fractions of B. malayi protein expressed
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 Vegetable peptone (or Tryptone) SOB + 20 mM Glucose lac promoter. Thus DE3 strains may exhibit uninduced target protein expres- sion. 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. T7 Express strains do not carry the DE3 prophage and bet- ter tolerate an SOS response.
Protease Deficient ([lon] ompT): E. coli B strains are "naturally" deficient in Antibiotics for Plasmid Selection
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 af- fect the transformation or growth characteristics of the cell.
Notice to Buyer/User: The buyer and user have a nonexclusive license to use this system or any
Genotype: fhuA2 [lon] ompT gal (
component thereof for RESEARCH PURPOSES ONLY. See Assurance Letter and Statement attached
λ DE3) [dcm] ∆hsdS hereto for details on terms of the license granted hereunder.
λ DE3 = λ sBamHIo ∆EcoRI-B int::(lacI::PlacUV5::T7 gene1) i21
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