Not all competent cells are equivalent with respect to molecular cloning applications. When choosing and preparing bacterial cells for transformation, factors that should be considered are the intended transformation procedure, including the cell’s genetic background, transformation efficiency, and growth rate; the throughput required; and notably, the researcher’s goals. These factors will directly impact the time and effort necessary for transformation experiments, as well as their success.
Transformation method highlights
The method of transformation to be used is one of the most important factors in choosing competent cells, because cells are prepared differently depending on whether they will be heat-shocked or electroporated (see competent cell preparation). The choice between the two methods, heat shock and electroporation, depends on transformation efficiency appropriate for the experimental goals, the complexity and quantity of the DNA to be transformed, and available resources (Table 1).
As noted in Table 1, the two methods each have advantages and challenges [1,2]. 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.
Table 1. Comparison of chemical transformation and electroporation.
| Chemical transformation (heat shock) | Electroporation | |
|---|---|---|
| Setup | No special equipment required | Special equipment required (e.g., electroporator) |
| Protocol | Well established | May vary by cell type |
| Transformation efficiency | 1 x 106 to 5 x 109 CFU/µg | 1 x 1010 to 3 x 1010 CFU/µg |
| Common research applications | Routine cloning and subcloning, protein expression | cDNA and gDNA library construction, transformation with low quantities of plasmid (e.g., pg) and large DNA (e.g., >30 kb) |
| Low to high; adaptable to high-throughput automation | Low to medium; may have limitations with high-throughput applications | |
| Compatible cell types | Limited range of bacterial species | Broader range of bacterial and other microbial species, including those with cell walls |
On the other hand, electroporation tends to be more efficient than heat shock. Hence, this method is amenable to a broader range of DNA amounts (from low to saturating concentrations), fragment sizes, and complexities. Cells that cannot be made chemically competent are also good candidates for electroporation, since electrocompetency stems from transient membrane polarization as a result of brief exposure to a high-voltage electric field (see electroporation). Caveats of electroporation include requirements for special equipment such as an electroporator and electroporation cuvettes, and potential protocol optimizations for electroporation of each bacterial strain.
Transformation efficiency and cloning applications
Transformation efficiency reflects the amount of supercoiled plasmid taken up by the competent cells; therefore, it 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.
For most cloning applications, a transformation efficiency between 106 and 1010 CFU/µg is considered adequate. Lower transformation efficiencies of approximately 106 CFU/µg can work well for routine cloning and subcloning experiments with supercoiled plasmids. Competent cells with higher transformation efficiencies (~108–109 CFU/µg) are desirable for transformation with more challenging DNA, such as ligations of blunt ends, short inserts, and low-input fragments. Electrocompetent cells with transformation efficiencies of >1 x 1010 CFU/µg are recommended for the most challenging samples such as gDNA and cDNA libraries, and plasmids larger than >30 kb (Figure 1).
Figure 1. Recommended ranges of transformation efficiency for common cloning applications.
Bacterial genotypes
A bacterial strain is defined as a subgroup of a bacterial species with specific genetic variations from the parental wild type. E. coli strains commonly used for transformation include DH5α, BL21, HB101, and JM109. Each strain can be described by its genotype, which lists mutations such as insertions and deletions (Figure 2). The genotype of a strain is critical in determining whether it can be used for the desired cloning applications.
The functions and phenotypes of genes are sometimes suggested by their three-letter symbols in the genotype. Related genes are distinguished by an uppercase letter following the three-letter code, e.g., lacY and lacZ for two of the genes of the lac operon. Phenotypic identifications are sometimes included in the genotype as unitalicized letters starting with a capital letter (e.g., TetR for tetracycline resistance of the INV110 strain; see Figure 2).
Figure 2. Genotype of bacterial strain INV110.
Table 2 describes genetic markers in the genotypes of common competent cells, their roles, and their benefits in transformation experiments. These genetic markers should be assessed when selecting competent cells to ensure their compatibility with research goals.
Table 2. Example genetic markers of E. coli strains commonly used in transformation.
| Genetic marker/ genotype | Wild-type gene’s function | Mutated gene’s phenotype or benefit |
|---|---|---|
| Cell growth | ||
| tonA/fhuA (cells are often labeled T1R) | Acts as a receptor for attachment of bacteriophages T1, T5, and f80 | Safeguards against bacterial cell infection and lysis by these bacteriophages (Figure 3A) |
| Colony screening | ||
| lacZΔM15 | Expresses 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 (Figure 3B) |
| DNA methylation | ||
| dcm/dam | Methylates C and A nucleotides of specific DNA sequences | Enables restriction of propagated plasmids by some methylation-sensitive enzymes |
| hsdRMS | Encodes R (restriction), M (modification/methylation), and S (specificity) subunits of endonucleases that recognize the EcoKI site | Enables propagation of unmethylated non–E. coli DNA (e.g., PCR amplicons) |
| mcrA, mcrBC, and mrr | Cleave certain sequences containing methylated C and A nucleotides (the sequences are distinct from the dam, dcm, EcoKI and EcoBI sites) | Permit propagation of methylated DNA of plant and animal origin |
| Plasmid purification | ||
| endA | Cleaves DNA nonspecifically | Improves yield and quality of plasmid DNA in purification (Figure 3C) |
| recA | Recombines homologous DNA sequences | Increases the stability of cloned plasmids carrying direct-repeat sequences Helps prevent recombination between plasmid DNA and host gDNA |
| Protein expression | ||
| lacIq | Overproduces the repressor of the lac operon promoter | Enables 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 infection | Enables ssDNA production |
More information about E. coli strains, genotypes, and genetic markers can be found in references 3 and 4.
Figure 3. Examples of bacterial genetic markers and their impacts.(A)tonA prevents infection by T1 bacteriophage (colored green). (B)lacZΔM15 enables screening of positive (white) and negative (blue) colonies. (C)endA prevents degradation of plasmid (manifested as gel smears) during isolation.
Bacterial growth rate
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 amounts of plasmid in a shorter time, accelerating the cloning workflow.
Figure 4 shows the growth rates of a number of bacterial strains and illustrates the potential time savings of using a fast-growing strain. Some fast strains form colonies within 8 hours of plating, allowing plating and picking of colonies on the same day. Similarly, some strains reach the plateau phase of the growth curve 4 hours after inoculation, allowing you to perform plasmid isolation sooner.
Figure 4. (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.
Experimental throughput considerations
The number of transformation reactions to be performed could 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 automation workflow.
In contrast, heat shock of chemically competent cells offers a more flexible setup for different throughputs (Figure 5). For low-throughput experiments, cells in individual tubes may be transformed with DNA by heat-shocking directly and conveniently, ensuring no loss of transformation efficiency from repeated freeze/thaw cycles. A similar approach may be considered for medium-throughput cloning, using a multichannel pipettor and cells in strip tubes. For high-throughput applications, 96-well formats allow multichannel pipetting, block incubation, and even automation, if needed.
Figure 5. Aliquots or packaging formats of chemically competent cells for low, medium and high throughputs.
Competent cells in medium- and high-throughput formats
Learn about different formats of competent cells for medium- and high-throughput applications, benefits of these formats, and how to use the flexible format in medium-throughput manual transformation.
Research goals and applications using competent cells
Successful propagation of different DNA types is often dictated by specific transformation efficiency, transformation methods, and bacterial genotypes. Examples of such DNA types include methylated DNA, large plasmids, phagemids, unstable constructs with repetitive sequences, DNA libraries, and expression vectors. Tips on how to select appropriate competent cells for common cloning applications are discussed in the next section.
In summary, properties of competent cells and their appropriate usage may significantly impact the success of cloning experiments. Understanding the differences among competent cells can facilitate planning of the transformation workflow and interpretation of colony screening for downstream research applications.
References
- Yoshida N, Sato M (2009) Plasmid uptake by bacteria: a comparison of methods and efficiencies. Appl Microbiol Biotechnol 83(5):791–798.
- Aune TE, Aachmann FL (2010) Methodologies to increase the transformation efficiencies and the range of bacteria that can be transformed. Appl Microbiol Biotechnol 85(5):1301–1313.
- E. coli Genetic Resources at Yale CGSC
- E. coli genotypes
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