Frequently Asked Questions - Bacterial Competent Cells

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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.

(cfu on control plate) / (ng of uncut vector) x (103 ng / µg) x (final dilution) = cfu / µg DNA

(Note: cfu = colony forming units)

Transformation efficiency refers to the number of cells that are transformed by 1µg DNA, usually a supercoiled plasmid.

These are cells that have been physically manipulated to increase their transformation efficiency.

Transformation is the process by which naked DNA is introduced into cells. It also can happen naturally at a low frequency.

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.

SOC added directly to the 1.5ml tube of competent cells followed by incubation at 300 rpm at 37°C will lower the transformation efficiency to about 40% of the standard efficiency.

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:

Ingredients Final Concentration
Yeast Extract 5g/l
Tryptone 20g/l
Sodium Chloride 10mM
Potassium Chloride 2.5mM
   
Autoclave, then add:  
Glucose 20mM
Magnesium Chloride 10mM
Magnesium Sulfate 10mM

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.

There is no difference from standard BL21. Some BL21 strains containing mutations will have slower growth.

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.

No. Protein levels depend on many factors, including the protein that is expressed.

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
lacUV5 promoter.
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.

These cells are ideal for high-level protein expression since they lack both ompT and Ion proteases.

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)

Genotype Advantage
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

These strains are the standard BL21 (DE3) strains initially developed by the Brookhaven National Laboratories. We have not introduced any additional modifications.

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.

Cells can be transformed either chemically or by electroporation. Chemical competency (ability to introduce DNA into cells) 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 "arcing" during the electrical shock. Both methods provide cells that can be frozen for storage.

(cfu on control plate) / (ng of supercoiled vector) x (103 ng / µg) x (final dilution) = cfu / µg DNA

(Note: cfu = colony forming units)

Transformation efficiency usually refers to the number of cells that are transformed by 1µg DNA, usually a supercoiled plasmid.

These are cells that have been physically manipulated to increase their transformation efficiency.

Transformation is the process by which naked DNA is introduced into cells. It also can happen naturally at a low frequency.

This does not directly apply since the cells are rinsed out of the cuvette with SOC before expression.

Yes, but anything less than 45 minutes can greatly reduce the number of colonies.

Standard SOC medium can be prepared as follows:

Ingredients Final Concentration
Yeast Extract 5g/l
Tryptone 20g/l
Sodium Chloride 10mM
Potassium Chloride 2.3mM
   
Autoclase, then add:  
Glucose 20mM
Magnesium Chloride 10mM
Magnesium Sulfate 10mM

Glucose and magnesium when present in SOC seem to improve transformation efficiency. Our transformation efficiencies are calculated using a standard 60-minute incubation with SOC after the transformation. Use of LB medium for recovery will result in reduced transformation efficiencies.

After bacteria have been transformed with an antibiotic-resistant plasmid, recovery in medium lacking the antibiotic allows for synthesis of the antibiotic resistance prior to plating them in the presence of antibiotic. The importance of recovery depends on the antibiotic used. For example, recovery is essential for kanamycin-resistant plasmids; whereas, ampicillin-resistant plasmid recovery improves the transformation efficiency 2-5 fold.

ElectroEB10B Competent Cells are a K12 derivative with similar growth. Generally, overnight incubation of plated transformed cells in LB medium and antibiotic at 37˚C results in medium size colonies (about 1mm in diameter). ElectroEB10B Competent Cells will grow faster than EB5Alpha (and other DH5α derivatives).

We calculate the transformation efficiency of our competent cells with 10pg supercoiled pUC19 and expect a transformation efficiency of at least 1x1010 colonies/µg pUC19 supercoiled DNA. Additionally the cells are tested with 10ng of pUC19 supercoiled DNA to insure that they can produce at least 1x108 colonies per 20µl electroporation

No, transformation efficiency, although proportional to the number of clones generated, is only one of many factors that will determine the number of clones after ligation of library construction. Other factors include: amount of vector used, quality of DNA, size of DNA, method of generating DNA (i.e. PCR, miniprep, etc.), restriction enzyme used (blunt or cohesive ends), systems used to generate clones (i.e. recombination or conventional ligation) and others. ElectroEB10B Competent Cells have been thoroughly controlled for quality and are guaranteed to exceed transformation efficiencies of 1010 colonies/µg with the pUC19 supercoiled DNA supplied.

Competent cell stability in terms of transformation efficiency is guaranteed by Edge BioSystems for at least 6 months, when properly stored at -80°C. Upon receipt of your cell shipment, cells should immediately be placed at -80°C to ensure optimal activity.

Freeze/thaw cycles will lower transformation efficiencies. If you want to use a tube for several transformations, we recommend quickly aliquoting the number of samples required and refreezing the tube in a dry ice:ethanol bath. Every time a tube is taken from -80°C storage conditions, the transformation efficiency is affected. Therefore, for optimal efficiencies a tube should not be reused.

Supercoiled DNA will transform at a 10-fold higher efficiency than cut/ligated DNA.

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. Before electroporation, the DNA should be diluted or precipitated as high DNA concentration in the electroporation can cause arcing.

Yes, ligated DNA does require dilution or desalting prior to transformation. For ligation reactions dilute at least 1:5 with water or TE buffer, or use a proven method for desalting before electroporation.

ElectroEB10B Competent Cells are best suited for applications that include a need for increased plasmid yield and quality, ultra efficient blue/white screening, generation of very large plasmid libraries and generating clones from methylated DNA. Edge BioSystems proudly offers its Ultra BL21 (DE3) and Ultra BL21 (DE3) pLysS Competent Cells, which have proven to be very successful in protein expression applications.

We recommend using 1µl DNA with 20µl cells for each transformation, larger size reactions can be used (up to 40µl in a 0.1cm gap cuvette) but the volume of DNA used should not be more than 5% of the cell volume.

These are satellite colonies. They are not transformants. Incubate your plates for less time (18 hours is good — never more than 20), use more antibiotic, or use fresher plates to get rid of them.

Because field strength is critical for bacteria, the choice of cuvette is important. In order to achieve the proper pulse, we suggest 1mm cuvettes. Yeast and fungi require 2mm cuvettes, while 4mm cuvettes are ideally suited to mammalian and human cells.

Avoiding conductive ions is a good way to avoid arcing when electroporating bacteria. This can be achieved by lowering the ratio of DNA-to-cells in the sample. Always avoid air bubbles and condensation with can accumulate on the electroporation cuvette. Dilute or precipitate ligation reactions before electroporation, carefully following washing procedures to remove salts. For ligation reactions dilute at least 1:5 with water or TE, or use a proven method for desalting before electroporation. Do not use more than 5µl total volume of DNA with 20µl cells. Using only highquality cuvettes can also reduce the chances of arcing.

Field strength is critical in electroporation. Field strength is usually expressed as kilovolts/centimeter, where kV is equal to the initial peak voltage and cm is equal to the size of the gap between the electrodes of the cuvette. Field strengths of greater than 15 kV/cm are usually recommended for bacteria.

This kit is intended for internal research use only by the purchaser. It is not to be used for human diagnostic purposes. In addition, "research use only" means that this kit and all of its contents are excluded from resale, repackaging or use for the making or selling of any commercial product or service without the written approval from Edge BioSystems.

ElectroEB10B Competent Cells offer distinct advantages in particular applications. Blue/white screening and high transformation efficiencies make them ideal for all demanding cloning applications and generation of very large plasmid libraries. Their genetic background provide excellent plasmid yield and quality with peace of mind for phage T1 and T5 resistance. The high efficiency also allows the cloning of limiting amounts of DNA. ElectroEB10B Competent Cells also can transform larger clones and clones containing methylated DNA.

Edge BioSystems guarantees >1010 colonies / µg pUC19. As with any other competent cell, the transformation efficiency will decrease with extremely large plasmids or ligated DNA.

Faster growing than EB5Alpha, these cells are ideal for demanding cloning needs and for the generation of large plasmid-based libraries and cloning methylated DNA. Their high efficiency makes them the cells of choice for cloning experiments using limiting amounts of DNA, especially since it tolerates ligated genomic DNA. Blue/white color selection facilitates screening of positive clones. ElectroEB10B Competent Cells are able to transform larger sized inserts allowing the construction of BAC and P1 clones.

ElectroEB10B: F- mcrA Δ(mmr-hsdRMS-mcrBC) Φ80lacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara, leu)7697 galU galK λ-rpsL nupG tonA.

Genotype Advantage
lacZΔM15 Allows blue/white screening through β-galactosidase complementation.
recA1 Eliminates general DNA recombination.
endA1 Improves yield and quality of DNA in plasmid preparations.
mcrA, mcrBC, mmr Allows cloning of methylated DNA including mammalian and plant genomic DNA.
hsdRMS Construction of more representative genomic libraries.
tonA Confers resistance to bacteriophages T1 and T5.

Different strains are clearly labeled as such. While we do not label the tube containing the product, there are other distinguishing labels on the packaging that make identification easy.

(cfu on control plate) / (ng of supercoiled vector) x (103ng / µg) x (final dilution) = cfu / µg DNA

(Note: cfu = colony forming units)

Transformation efficiency usually refers to the number of cells that are transformed by 1µg DNA, usually a supercoiled plasmid.

These are cells that have been physically manipulated to increase their transformation efficiency

Transformation is the process by which naked DNA is introduced into cells. It also can happen naturally at a low frequency

Yes, you can add 250µl SOC directly to the 1.5ml transformation tube and shake it at 300 rpm for 1 hour without altering the transformation efficiency.

The following table shows recovery done with 1ml SOC in 14ml round bottom culture tubes. The percentages are relative to 60 minutes of recovery:

Minutes
Transformation efficiency using pET41 plating in LB/Kan
Transformation efficiency using pUC19 plating in LB/amp
 
 
 
60
100
100
45
76
87
30
19
60
15
2
63
0
0
59

Standard SOC medium can be prepared as follows:

Ingredients Final Concentration
Yeast Extract 5g/l
Tryptone 20g/l
Sodium Chloride 10mM
Potassium Chloride 2.5mM
   
Autoclase, then add:  
Glucose 20mM
Magnesium Chloride 10mM
Magnesium Sulfate 10mM

Glucose and magnesium present in SOC seem to improve transformation efficiency. Our transformation efficiencies are calculated using a standard 60-minute incubation with SOC after the transformation.

After bacteria have been transformed with an antibiotic-resistant plasmid, recovery in medium lacking the antibiotic allows for synthesis of the antibiotic resistance prior to plating them in the presence of antibiotic. The importance of recovery depends on the antibiotic used. For example, recovery is essential for kanamycin-resistant plasmids; whereas, ampicillin-resistant plasmid recovery improves the transformation efficiency 2-5 fold.

EB5Alpha Competent Cells is a K12 derivative with similar growth. Generally, overnight incubation of plated transformed cells in LB/antibiotic at 37˚C results in medium size colonies (about 1mm in diameter).

We calculate the transformation efficiency of our competent cells with 10pg supercoiled pUC19 and expect a transformation efficiency of at least 2x109 colonies/µg pUC19 supercoiled DNA.

No, transformation efficiency, although proportional to the number of clones generated, is only one of many factors that will determine the number of clones after ligation of library construction. Other factors include: amount of vector used, quality of DNA, size of DNA, method of generating DNA (i.e. PCR, miniprep, etc.), restriction enzyme used (blunt or cohesive ends), systems used to generate clones (i.e. recombination or conventional ligation) and others. EB5Alpha Competent Cells have been thoroughly controlled for quality and are guaranteed to exceed transformation efficiencies of 109 colonies/µg pUC19 supercoiled DNA.

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.

We compared transformation efficiencies of supercoiled pUC19 vs. ligated pUC19 after single and double cut. We also tested if heat activation of the ligase improved transformation efficiency. The results are shown below. Please note that in order to make the experiments comparable, transformations were not done according to our standard protocols. Transformation efficiencies with standard protocols exceed 109cfu/µg pUC19.

Material (pUC19) Transformation Efficiency % Efficiency Relative to Supercoiled pUC19
Supercoiled pUC19 5 x 108 100
Single cut pUC19, heat inactivated ligation 6.2 x 108 100
Single cut pUC19 5.4 x 108 100
Double cut pUC19, heat inactivated ligation 5.4 x 107 10.8
Double cut pUC19 3.9 x 107 7.8

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.

EB5Alpha Competent Cells are best suited for applications that include a need for increased plasmid yield and quality, ultra efficient blue/white screening for cloning and generation of cDNA libraries. Although EB5Alpha cells can be used for protein expression, Edge BioSystems proudly offers its Ultra BL21 (DE3) and Ultra BL21 (DE3) pLysS Competent Cells, which have proven to be very successful in protein expression applications.

This kit is intended for internal research use only by the purchaser. It is not to be used for human diagnostic purposes. In addition, "research use only" means that this kit and all of its contents are excluded from resale, repackaging or use for the making or selling of any commercial product or service without the written approval from Edge BioSystems.

EB5Alpha Competent Cells offers distinct advantages in particular applications. Blue/white screening and high transformation efficiencies make them ideal for all cloning applications and generation of libraries. They are especially suitable for high-throughput cloning applications. Their genetic background provide excellent plasmid yield and quality with peace of mind for phage T1 and T5 resistance.

Edge BioSystems guarantees >109 colonies / µg pUC19. As with any other competent cell, the transformation efficiency will decrease with larger plasmids or ligated DNA.

These cells are ideal for most cloning needs and for the generation of plasmid-based libraries. Their high efficiency makes them the cells of choice for cloning experiments using limiting amounts of DNA in either a high-throughput or single use format. Blue/white color selection facilitates screening of positive clones.

EB5Alpha:   F-Φ80lacZΔM15 Δ(lacZYA-argF)U169 recA1 endA1 hsdR17(rk-,mk+) phoA supE44 thi-1 gyrA relA1 tonA

Genotype Advantage
lacZΔM15 Allows blue/white screening through β-galactosidase complementation.
recA1 Eliminates general DNA recombination.
endA1 Improves yield and quality of DNA in plasmid preparations.
hsdR17(rk-,mk+) Represents restriction negative and modification positive phenotype and allows cloning of DNA without cleavage by endogenous restriction endonucleases.
relA1 Permits RNA synthesis in the absence of protein synthesis.
tonA Confers resistance to bacteriophages T1 and T5.

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.

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. Before electroporation, the DNA should be diluted or precipitated as high DNA concentration in the electroporation can cause arcing.

Yes, ligated DNA does require dilution or desalting prior to transformation. For ligation reactions dilute at least 1:5 with water or TE buffer, or use a proven method for desalting before electroporation.

We recommend using 1µl DNA with 20µl cells for each transformation, larger size reactions can be used (up to 40µl in a 0.1cm gap cuvette) but the volume of DNA used should not be more than 5% of the cell volume.

These are satellite colonies. They are not transformants. Incubate your plates for less time (18 hours is good — never more than 20), use more antibiotic, or use fresher plates to get rid of them.

Because field strength is critical for bacteria, the choice of cuvette is important. In order to achieve the proper pulse, we suggest 1mm cuvettes. Yeast and fungi require 2mm cuvettes, while 4mm cuvettes are ideally suited to mammalian and human cells.

Avoiding conductive ions is a good way to avoid arcing when electroporating bacteria. This can be achieved by lowering the ratio of DNA-to-cells in the sample. Always avoid air bubbles and condensation that can accumulate on the electroporation cuvette. Dilute or precipitate ligation reactions before electroporation, carefully following washing procedures to remove salts. For ligation reactions dilute at least 1:5 with water or TE, or use a proven method for desalting before electroporation. Do not use more than 5µl total volume of DNA with 20µl cells. Using only highquality cuvettes can also reduce the chances of arcing.

Field strength is critical in electroporation. Field strength is usually expressed as kilovolts/centimeter (kV/cm), where kV is equal to the initial peak voltage and cm is equal to the size of the gap between the electrodes of the cuvette.

Proteins toxic to E.coli are better expressed in Acella™ Chemically Competent Cells 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 Acella™ Electrocompetent Cells and a T7 lac promoter.

Our Acella™ Electrocompetent Cells are designed for high-level protein expression using T7 RNA polymerase-based expression systems. Acella™ pLysS Chemically Competent Cells provides tighter control for expression of toxic proteins. Tighter control is provided by strains carrying the pLysS plasmids encoding T7 lysozyme, a natural inhibitor of T7 RNA polymerase.

The expression properties of Acella™ Electrocompetent Cells are the same as the standard BL21. It is difficult to predict because 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. With the exception of the added ΔendA ΔrecA genotype, the Acella™ Electrocompetent Cells are identical to standard BL21. BL21 cells naturally lack ompT and Lon proteases improving stability of synthesized proteins. BL21 (DE3) expresses T7 polymerase after induction with IPTG, therefore regulating the transcription of genes under the T7 promoter (for example, those in pET plasmids).

Yes. Acella™ Electrocompetent Cells have transformation efficiencies exceeding 1010 , 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 a lower transformation efficiency. You are thereby able to directly transform cDNA libraries into Acella™ Electrocompetent Cells and readily purify the DNA. In fact, high transformation efficiencies make Acella™ suitable for applications that include a need for increased plasmid yield and quality and generation of large plasmid libraries. The complete deletion of endA and recA genes makes this strain ideal for cloning purposes and DNA isolation. Since Acella™ Electrocompetent Cells are a fast growing strain, miniprep cultures can be grown for 3-4 hours and plasmids can be analyzed the day after the transformation, saving one extra day. Positive clones can then be grown directly for protein expression.

The following chart can be used as a general guideline:

Electrocompetent Cell Strain Description Application
Acella™ (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 lacUV5 promoter. Recombinant protein production non-toxic to E. coli, high quality plasmid preparation with short culture time (3 hours) for plasmid miniprep.

Edge BioSystems provides these materials for research purposes only. Acella™ Electrocompetent 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).

Many 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. Some proteins require post-translational modifications in order to be completely functional and E. coli cells do not perform these modifications. 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 of the protein.

Edge BioSystems guarantees >1010 colonies / µg pUC19. As with any other electrocompetent cell, the transformation efficiency may decrease with larger plasmids or ligated DNA.

Their high transformation efficiency and ∆endArecA genotype make them ideal as a standard cloning strain with high plasmid yield and their BL21 background makes them a fast growing strain. These cells are ideal for high-level protein expression since they lack both ompT and Ion proteases. Acella™ Electrocompetent Cells are particularly effective in directly transforming cDNA libraries with an expression vector. Acella™ Electrocompetent Cells can be used for cloning and expression purposes in just one transformation step and they have the advantage of fast growth, making it possible to isolate miniprep plasmid DNA after only three hours of culture. All of these features make it possible to save a total of two to three days of work and eliminate the need for additional cloning strains and reagents.

Genotype: F-ompT hsdSB(rB-mB-) gal dcm (DE3) ΔendA ΔrecA

Genotype Advantage
DE3 A lambda derivative bacteriophage that carries the gene for T7 RNA polymerase under the control of the lacUV5 promoter (inducible by IPTG)
ompT Deficiency 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
ΔendA Complete deletion of endA gene (DNA specific endonuclease I) shown to improve yield and quality of DNA from plasmid minipreps.
ΔrecA Complete deletion of recA gene (gene central to general recombination and DNA repair) shown to eliminate general recombination and render bacteria sensitive to UV light.

 

These strains are derivatives from the standard BL21 (DE3) strains initially developed by the Brookhaven National Laboratories. We deleted the endA and recA genes without introducing additional mutations or antibiotic resistance. The only difference between the standard BL21 (DE3) strains developed by the Brookhaven National Laboratories and our Acella™ strain is the complete deletion of those two genes.

Different strains are clearly labeled as such on the box. Please note that tubes of cells are not individually labeled.

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 "arcing" during the electrical shock. Both methods provide cells that can be frozen for storage.

[ (cfu on control plate) / (pg of supercoiled pUC19) ] x (106 pg / µg) x (final dilution) = cfu / µg DNA

(Note: cfu = colony forming units)

Transformation efficiency refers to the number of cells that are transformed by 1µg DNA, usually a supercoiled plasmid.

These are cells that have been physically manipulated to increase their transformation efficiency.

Transformation is the process by which naked DNA is introduced into cells. It also can happen naturally at a low frequency.

We have tested the transformation efficiency of our 96-Well 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.

1 ml SOC added directly to the 1.5ml tube of competent cells followed by incubation at 300 rpm at 37°C will lower the transformation efficiency.

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:

Ingredients Final Concentration
Yeast Extract 5g/l
Tryptone 20g/l
Sodium Chloride 10mM
Potassium Chloride 2.5mM
   
Autoclave, then add:  
Glucose 20mM
Magnesium Chloride 10mM
Magnesium Sulfate 10mM

 

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. Incubation in other medium, different from SOC during recovery may result in lower transformation efficiencies.

Acella™ Chemically Competent Cells grow at the same rate in liquid medium and they grow slightly slower than standard BL21 cells in agar plates. Growth of Acella™ Chemically Competent Cells is several times faster than K-12 derivative cloning strains in liquid and in agar medium.

We calculate the transformation efficiency of our competent cells with 10pg supercoiled pUC19 and expect a transformation efficiency of at least 2x108 colonies/µg pUC19 supercoiled DNA. We test the presence of the pLysS plasmid in Acella™ pLysS Chemically Competent Cells by plating them in the presence of chloramphenicol.

No. Protein levels depend on many factors, including the protein that is expressed.

No, transformation efficiency, although proportional to the number of clones generated, is only one of many factors that will determine the number of clones after ligation of library construction. Other factors include: Amount of vector used, quality of DNA, size of DNA, method of generating DNA (i.e. PCR, miniprep, etc.), restriction enzyme used (blunt or cohesive ends), systems used to generate clones (i.e. recombination or conventional ligation) and others. Acella™ Chemically Competent Cells have been thoroughly controlled for quality and are guaranteed to exceed transformation efficiencies of 2x108 colonies/µg with the pUC19 supercoiled DNA supplied.

Competent cell stability in terms of transformation efficiency is guaranteed by Edge BioSystems for at least 12 months, when properly stored at -80°C. Upon receipt of your cell shipment, cells should immediately be placed at -80°C to ensure optimal activity.

Acella™ Chemically 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.

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 Acella™ Chemically Competent Cells 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 an Acella™ pLysS Chemically Competent Cell strain and a T7 lac promoter.

Acella™ Chemically Competent Cells strains are designed for high-level protein expression using T7 RNA polymerasebased expression systems. Acella™ pLysS Chemically Competent Cells strains provides tighter control for expression of toxic proteins. Tighter control is provided by strains carrying the pLysS plasmids encoding T7 lysozyme, a natural inhibitor of T7 RNA polymerase.

The expression properties of Acella™ Chemically Competent Cells strains are the same as the standard BL21. It is difficult to predict because 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, with the exception of the added ΔendA ΔrecA genotype, the Acella™ Chemically Competent Cells are identical to standard BL21 (DE3). BL21 cells naturally lack ompT and Lon 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' Acella™ Chemically Competent Cells strains have a transformation efficiency of >2 x 108, usually as high as 5 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 complete deletion of endA and recA genes makes this strain ideal for cloning purposes and DNA isolation. Since Acella™ Chemically Competent Cells are a fast growing strain, miniprep cultures can be grown for 3-4 hours and plasmids can

The following chart can be used as a general guideline:

Chemically Competent Cell Strain Description Application
Acella™ (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 lacUV5 promoter. Recombinant protein production non-toxic to E. coli, high quality plasmid preparation with short culture time (3 hours) for plasmid miniprep.
Acella™ 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. Recombinant protein production that may be toxic to E. coli or lower 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. Note that the pLysS plasmid may be present in plasmids preparations from this strain.

Edge BioSystems provides these materials for research purposes only. Acella™ Chemically 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 when expressed in E. coli cells. Some proteins require post-translational modifications in order to be completely functional and E. coli cells do not perform these modifications. 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 of the protein.

Edge BioSystems guarantees >2 x 108 colonies / µg pUC19, although transformation efficiencies are usually about 5 x 108 colonies / µg pUC19. As with any other competent cell, the transformation efficiency will decrease with larger plasmids or ligated DNA.

Their high transformation efficiency and ΔendA ΔrecA genotype make them ideal as a standard cloning strain with high plasmid yield and their BL21 background makes them a fast growing strain. These cells are ideal for high-level protein expression since they lack both ompT and Ion proteases. Acella™ Chemically Competent Cells can be used for cloning and expression purposes in just one transformation step and they have the advantage of fast growth, making it possible to isolate plasmid DNA after only three hours of miniprep culture. All these features make it possible to save a total of two to three days of work and eliminate the need for additional cloning strains and reagents.

Acella™ Chemically Competent Cells: F-ompT hsdSB (rB-mB-) gal dcm (DE3) ΔendA &Detla;recA
Acella™ pLysS Chemically Competent Cells: F-ompT hsdSB (rB- mB-) gal dcm (DE3) ΔendA ΔrecA pLysS (CamR)

Genotype Advantage
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 plasmin.
ompT Deficiency 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
ΔendA Complete deletion of endA gene (DNA specific endonuclease I) shown to improve yield and quality of DNA from plasmid minipreps.
ΔrecA Complete deletion of recA gene (gene central to general recombination and DNA repair) shown to eliminate general recombination and render bacteria sensitive to UV light.

 

These strains are derivatives from the standard BL21 (DE3) strains initially developed by the Brookhaven National Laboratories. We deleted the endA and recA genes without introducing additional mutations or antibiotic resistance. The only difference between the standard BL21 (DE3) strains developed by the Brookhaven National Laboratories and our Acella™ strain is the complete deletion of those two genes.

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.