Six Considerations for Competent Cell Selection - US (2024)

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When selecting and preparing bacterial cells for transformation, several factors should be considered. These considerations will directly impact the time and effort necessary for transformation experiments, as well as the outcomes.

The six key considerations to focus:

  1. Selecting the right transformation method
  2. Optimizing transformation efficiency
  3. Choosing the right bacterial genotype
  1. Managing bacterial growth rate and cell density
  2. Adapting to your experimental throughput needs
  3. Aligning to your research goals

Selecting the right transformation method: electroporation vs. chemical transformation

The transformation method used is one of the primary factors in selecting competent cells. Depending on whether the cells will undergo heat shock or electroporation, the method of competent cell preparation differs. The choice between the two methods is determined by several factors. These include the transformation efficiency appropriate for the experimental design, the size and quantity of the DNA to be transformed, and the available equipment. Table 1 lists the chemical transformation and electroporation features [1,2].

Table 1. Comparison of chemical transformation and electroporation

Chemical transformation (heat shock)Electroporation
SetupOnly standard equipment needed (water bath or dry bath/block heater)Requires specialized equipment (electroporator and electroporation cuvettes)
ProtocolLonger, but less prone to errorsStandardized, but more sensitive
Transformation efficiency1 x 106 to 5 x 109 CFU/µg1 x 1010 to 3 x 1010 CFU/µg
Common research applicationsRoutine cloning and subcloning, protein expressioncDNA and gDNA library construction, transformation with low quantities of plasmid (e.g., pg) and large DNA (e.g., >30 kb)

Throughput

Low to high; adaptable to high-throughputcloning workflow, competent cells available in various formatsLow to medium; may have limitations with high-throughput applications
Compatible cell typesLimited range of bacterial speciesBroader range of bacterial and other microbial species, including those with cell walls

Chemical transformation or heat shock can be performed in a simple lab setup, yielding transformation efficiencies that are usually sufficient for routine cloning and subcloning applications. Since the cell membrane is made more permeable by cation treatment and heat shock, certain cell types, such as those with cell walls, may not be favorable to chemical transformation.

Electroporation tends to be more efficient than heat shock and is amenable to a broader range of DNA amounts, from low to saturating concentrations and varying fragment sizes. Cells that are unsuitable for chemical competency are good candidates for electroporation, since electro competency stems from transient membrane polarization resulting from brief exposure to a high-voltage electric field. Specialized equipment Is required for electroporation (some instruments can be used only for eucaryotic or procaryotic cells) and potentially the transformation protocol might need additional optimizations for the cell strain used.

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Optimizing transformation efficiency

Transformation efficiency reflects the amount of supercoiled plasmid taken up by the competent cells; therefore, it directly impacts the cloning efficiency, which is a measure of overall success to obtain clones with the desired plasmid. Competent cells may display varying efficiencies of transformation, depending on the method of cell preparation, storage, the type of transforming DNA, and other factors.

What is a good transformation efficiency?

Guidelines on transformation efficiencies are listed below.

  • Transformation efficiencies between 106 and 1010 CFU/µg is considered adequate for most cloning applications.
  • Lower transformation efficiencies of approximately 106 CFU/µg can work well for routine cloning and subcloning experiments with supercoiled plasmids.
  • Higher transformation efficiencies of ~108–109 CFU/µg are desirable for more difficult to clone constructions, such as blunt-end ligations, short or large inserts, and low-input fragments.
  • To achieve transformation efficiencies greater than 1 x 1010 CFU/µg, electrocompetent cells are recommended. Electrocompetent cells are suitable for challenging samples such as gDNA and cDNA libraries, and plasmids larger than 30 kb or cloning using limited quantities of DNA (e.g., 10 pg) (Figure 1).
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Figure 1. Recommended ranges of transformation efficiency for common cloning applications.

How to calculate transformation efficiency

To calculate the transformation efficiency, divide the number of transformants by the amount of DNA added, and factor in cell dilution (if performed), using the following formula:


With ligated DNA, the amount of DNA added to the cells can also be determined from the ligation reaction setup, DNA dilution (if performed), and DNA volume for transformation, using the following formula:

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Example calculations of transformation efficiency

50 ng of DNA is ligated in a 20 μL reaction. After ligation, the reaction is diluted 2-fold and 5 μL of the diluted ligation mixture is added to 100 μL of competent cells for transformation.

DNA added to cells = (0.05 µg/20 µL) x 1/2 x 5 µL = 0.00625 µg

After transformation, the cell suspension is diluted 5-fold and 200 µL of the diluted cells are plated. 300 colonies are formed after overnight incubation.

Transformation efficiency = (300 CFU/0.00625 µg) x (100 µL/200 µL) x 5 = 1.2 x 105 CFU/µg

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Choosing the right bacterial genotype

The genotype of a strain is critical in determining whether it can be used for the desired cloning applications. Genetic markers of competent cells should be assessed when selecting competent cells to ensure their compatibility with research goals. Table 2 describes genetic markers in the genotypes of common competent cells, their roles, and their benefits in transformation experiments.

Table 2. Example genetic markers of E. coli strains commonly used in transformation

Genetic marker/ genotypeWild-type gene functionMutated gene phenotype or benefit
Cell growth
tonA (also known as fhuA)
(cells are often labeled T1R)
Acts as a receptor for attachment of bacteriophages T1, T5, and φ80Safeguards against bacterial cell infection and lysis by bacteriophages
Colony screening
lacZΔM15Expresses a mutant lacZ gene, which can be complemented with the alpha peptide of beta-galactosidase (alpha complementation)Enables identification of desired clones by blue/white colony screening
DNA methylation
dcm/damMethylates C and A nucleotides of specific DNA sequencesEnables restriction of propagated plasmids by some methylation-sensitive enzymes
hsdRMSEncodes R (restriction), M (modification/methylation), and S (specificity) subunits of endonucleases that recognize the Eco*kI siteEnables propagation of unmethylated non-E.coli DNA (e.g., PCR amplicons)
mcrA, mcrBC, and mrrCleave certain sequences containing methylated C and A nucleotides (the sequences are distinct from the dam, dcm, Eco*kI, and EcoBI sites)Permit propagation of methylated DNA of plant and animal origin
Plasmid purification
endACleaves DNA nonspecificallyImproves yield and quality of plasmid DNA in purification
recARecombines hom*ologous DNA sequencesIncreases the stability of cloned plasmids carrying direct-repeat sequences
Helps prevent recombination between plasmid DNA and host gDNA
Protein expression
lacIqOverproduces the repressor of the lac operon promoterEnables tight regulation of transcription of the lac operon with IPTG
Propagation of single-stranded DNA (ssDNA)
F′Encodes strand-like structures called F pili on the outer membrane of E. coli that allow M13 phage infectionEnables ssDNA production

More information about E. coli strains, genotypes, and genetic markers can be found in references 3 and 4.

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Bacterial growth rate and cell density

The growth rate or doubling time of a bacterial strain is another consideration in selecting competent cells. Faster-growing cells form colonies and produce sufficient plasmids in a shorter time, accelerating the cloning workflow.

Figure 2 shows the growth rates of multiple bacterial strains and illustrates the potential time savings of using a fast-growing strain. For example, Mach 1 T1Rform colonies within 8 hours of plating, allowing plating and picking of colonies on the same day. As well, this strain reaches the plateau phase of the growth curve four hours after inoculation, allowing you to perform plasmid isolation sooner.

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Figure 2. Bacterial growth rate and potential time savings of different bacterial strains. (A) Growth rates of five different bacterial strains, as measured by OD600. (B) Time comparisons for a standard bacterial strain and a fast-growing bacterial strain to form colonies and produce plasmids.

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Adapting to your experimental throughput needs

The number of transformation reactions to be performed may be a deciding factor in the choice of competent cells. When considering heat shock vs. electroporation, the latter may not be amenable to high-throughput cloning due to requirements for an electroporator and cuvettes, as well as possible challenges with setting up an automated workflow. In contrast, heat shock of chemically competent cells allows flexible setup for different throughputs (Figure 3).

  • For low-throughput experiments, cells in individual tubes (One Shot format) may be transformed with DNA by applying heat shock directly and conveniently, ensuring no loss of transformation efficiency from repeated freeze/thaw cycles.
  • For high-throughput applicationsseveral MultiShot formats can be selected with varying flexibility (e.g., ability to separate one or more tubes from the strip of tubes (StripWell and FlexPlate) or PCR 96-well plates for automated workflows).

A. OneShot format

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B. MultiShot stripwell format

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C. MultiShot 96-well plate

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Figure 3. Aliquots or packaging formats of chemically competent cells from low- to high-throughputs.

Watch video on the various formats ›

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Aligning to your research goals

To select competent cells for your experiments, it’s important to make sure your choices align with your research goals. Your research may involve various DNA constructs, includingmethylated DNA, large plasmids, phagemids, unstable constructs with repetitive sequences, DNA libraries, and expression vectors.Choosing competent cells that can be successful recipients of target DNA constructs is crucial to ensuring the desired transformation outcome.

Additionally, the propagation of different target DNA constructs is also determined by transformation efficiency, transformation methods, and bacterial genotypes.

Learn more on how to select appropriate competent cells for common cloning applications

In summary, properties of competent cells and their appropriate usage may significantly impact the success of cloning experiments. Understanding the differences among competent cells enables planning of thetransformation workflow and interpretation of colony screening for downstream research applications.

References

Resources

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