Great turn around time on my 80 exome project!
Cells can be made competent either chemically or by electroporation. Chemical competency usually involves treatment with divalent cations at low temperatures, followed by a quick cold-heat transfer during transformation. Electroporation involves the removal of salts that may cause "arching" during the electrical shock. Both methods provide cells that can be frozen for storage.
We have tested the transformation efficiency of our plates using a thermocycler with a ramp of 1.5°C per second (with actual times of 28 seconds from 0°C to 42°C and 110 seconds from 42°C to 0°C) and the transformation efficiency was 40% of the standard efficiency. Using 2 thermocyclers and transferring the plate by hand, the transformation efficiency was 60% of the standard efficiency. In both cases, the cells were recovered in 0.5ml of SOC in a 2ml deep well plate at 300 rpm, 37°C.
When transforming with an ampicillin-based plasmid, 15-30 minutes of recovery will give about 70% of the standard efficiency. For kanamycin-based plasmids, 30 minutes of recovery will give about 50% of the standard efficiency, and 45 minutes recovery will give efficiency similar to 1 hour of recovery.
Standard SOC medium can be prepared as follows:
|Autoclave, then add:|
An incubation of 45-60 minutes with SOC lets bacteria recover after transformation. Our transformation efficiencies are calculated using a standard 60-minute incubation with SOC after the heat shock step. If SOC recovery is omitted, the transformation efficiency may decrease up to 10-fold for ampicillin-resistant plasmids and up to 100-fold for kanamycinresistant plasmids.
We calculate the transformation efficiency of our ultra competent cells with supercoiled pUC19 and test the presence of the pLysS plasmid in the case of Ultra BL21 (DE3) pLysS Competent Cells by plating them in the presence of chloramphenicol.
Competent cell stability in terms of transformation efficiency is guaranteed by Edge BioSystems for at least 3 months, when properly stored at -70°C. Upon receipt of your cell shipment, cells should immediately be placed at -70°C to ensure optimal activity.
Ultra BL21 (DE3) Competent Cells express the T7 polymerase under induction with IPTG. Plasmids containing the gene of interest under the control of a standard T7 promoter or a variant of it (for example, a T7-lac promoter) can be used with this system.
The optimal concentration of IPTG may vary from protein to protein. In other words, a concentration that works well for one protein may be too high for another, therefore resulting in insolubilization. In general, 0.4mM IPTG provides full induction of genes under the T7 promoter and 1mM is recommended for full induction of genes under the T7 lac promoter.
|Material||Transformation Efficiency||% Efficiency Relative to Supercoiled pUC19|
|Supercoiled pUC19||1.8 x 107||100|
|Single cut pUC19||6.8 x 106||37.8|
|Double cut pUC19||2.4 x 106||13.3|
Note: This experiment was done using cells with low transformation efficiency. Consequently, the efficiency is usually about ten fold higher.
The more DNA used, the more efficient the ligation will be. The ratio of vector to inserted DNA can be critical for obtaining high-efficiency ligations. A molar excess of insert to vector may yield higher efficiency ligations when subcloning inserts into plasmid vectors. However, an equal or greater ratio of vector to insert may be preferred when performing library construction into plasmid vectors.
Proteins toxic to E.coli are better expressed in BL21 containing the pLysS plasmid. The pLysS plasmid will express low levels of lysozyme that will bind to T7 polymerase, therefore inhibiting transcription. This will lower the basal expression of the protein during the pre-induction growth. If the protein is extremely toxic, then it is better expressed in a BL21 (DE3) pLysE strain or using a combination of a BL21 (DE3) pLysS strain and a T7 lac promoter.
Our Ultra BL21-derived Competent Cell strains are designed for high-level protein expression using T7 RNA polymerase-based expression systems. Our Ultra BL21 (DE3) pLysS Competent Cell strain provides tighter control for expression of toxic proteins.
That is difficult to predict. Protein levels as well as solubility will vary from protein to protein. In general, long proteins are much more difficult to express than shorter proteins. Also, human proteins that contain clusters of codons rarely used in E.coli may have a tendency to give lower yields and/or truncated products.
Yes, Ultra BL21 Competent Cells are identical to standard BL21. BL21 cells naturally lack ompT and Ion proteases improving stability of synthesized proteins. BL21 (DE3) expresses T7 polymerase after induction with IPTG, therefore promoting the transcription of genes under the T7 promoter (for example, those in pET plasmids).
Yes. Edge Biosystems' Ultra BL21 Competent Cells strains have a transformation efficiency of >2 x 108 making it possible to eliminate the intermediate step of cloning into a different strain of E.coli, purifying the DNA and then transforming into a BL21 strain with lower transformation efficiency.
The following chart can be used as a general guideline:
|Competent Cell Strain||Description||Application|
|BL21 (DE3)||(DE3) indicates that the host is a lysogen
of λDE3 and therefore carries a
chromosomal copy of the T7 RNA
polymerase gene under control of the
|Recombinant protein production nontoxic to E.coli|
|BL21 (DE3) pLysS||Coupled with the response above, the
addition of pLysS indicates plasmid
encoding small amounts of T7 lysozyme,
which is a natural inhibitor of T7 RNA
polymerase and lowers the basal levels of
recombinant protein before induction.
|In addition to the above information,
recombinant proteins can affect cell
growth and viability. In the presence
of detergents, small levels of
lysozyme will help break the cell wall,
which facilitate cell lysis once the
protein is already expressed.
The Ultra BL21 (DE3) pLysS Competent Cells are based on the T7 expression system. This is technology developed at Brookhaven National Laboratory under contract with the U.S. Department of Energy. Consequently, U.S. patents assigned to Brookhaven Science Associates (BSA) protect this technology.
These materials are to be used by noncommercial entities for research purposes only. Commercial entities require a license from BSA. You may refuse these cells by returning the enclosed materials unused.
To obtain information about licensing, please contact the Office of Intellectual Property and Partnerships, Brookhaven National Laboratory, Building 475D, Upton, NY 11973 (telephone: 631-344-7134 or fax: 631-344-3729).
Most recombinant proteins can be cloned and expressed in E.coli. The use of E.coli for protein expression is well documented for its advantages of low cost, easy transformation and fermentation, and high protein yields. However, solubility may be an issue, since some proteins are insoluble and aggregate in inclusion bodies. It is important to note that the following factors can affect expression levels and/or solubility: growing temperature, concentration of inducer (IPTG), host strain, protein size and structure, and toxicity.
Edge BioSystems guarantees >2 x 108 colonies / µg pUC19, although transformation efficiencies are usually higher. As with any other competent cell, the transformation efficiency will decrease with larger plasmids or ligated DNA.
Ultra BL21 (DE3): F-ompT hsdSB (rB-mB-) gal dcm (DE3)
Ultra BL21 (DE3) pLysS: F-ompT hsdSB (rB-mB-) gal dcm (DE3) pLysS (CamR)
|DE3||A lambda derivative bacteriophage that carries the gene for T7 RNA polymerase under the control of
the lacUV5 promoter (inducible by IPTG)
|pLysS||A plasmid that expresses low levels of T7 lysozyme and is a natural inhibitor of T7 polymerase. It
reduces basal levels of expression of recombinant genes and improves expression of toxic genes.
|CamR||This denotes chloramphenicol resistance and is a selection marker of pLysS plasmid. We
recommend a working concentration of 35 µg/ml for maintenance of pLysS plasmid.
|ompT||Defficiency in the ompT protease, which results in higher yields of intact recombinant proteins|
|hsdSB (rB-mB-)||Allows cloning of DNA without cleavage by endogenous restriction endonucleases|
Different strains are clearly labeled as such. While we do not label the aluminum seal affixed to the plate product, there are other distinguishing labels that make identification easy. Labeling the aluminum seal would prevent the piercing characteristic.