Choosing the Right Bug

TRANSFORMATION EFFICIENCY

Bacterial cells can be made competent by chemical treatment or by electroporation. Transformation by electroporation is easier: Just mix the DNA and cells in a specialized cuvette, apply electric current, and you're ready to go, although the technique requires that the necessary hardware be on hand. Chemical treatment by calcium chloride followed by heat shock is slightly more cumbersome but easier on the checkbook. The latter method often results in lower transformation efficiencies compared to electroporation (measured in colony-forming units per microgram of plasmid DNA, or cfu/μg).

Efficiencies between 1 × 10⁶ and 1 × 10⁹ cfu/μg are generally adequate for routine cloning and subcloning applications or when plenty of DNA is available. When DNA is limited, however, or when delivering large plasmids, electroporation-competent cells might be a better choice. With transformation efficiencies exceeding 1 × 10¹⁰ cfu/μg), these cells are also useful for constructing complex DNA libraries.

GENOTYPE

Choosing a competent cell strain can be quite complex. Some traits simplify identification of clones that have taken up the desired DNA. Often, cloning vectors confer resistance to one or more antibiotics such as ampicillin or tetracycline. The cells must be susceptible to a particular antibiotic, so that those with the vector can be selected easily. When trying to home in on cells that have taken up a particular fragment, some researchers employ strains that support alpha-complementation of the lacZ (beta-galactosidase) gene, which enables insertion-containing clones to be selected based on whether they are blue or white when grown on IPTG and X-gal media.

Many researchers elect to purchase recA mutant strains that are recombination-deficient. The recA repair system, which recombines homologous sequences, is disabled to increase clone stability. This mutation can hinder cell growth, however, so strains designed primarily for protein expression (e.g., BL21) do not include it.

Some DNA is particularly unstable, such as genes that exhibit secondary structure or contain long inverted repeats that may be deleted by E. coli DNA repair systems. To clone this type of DNA, researchers can obtain strains of competent cells that are deficient in systems such as UV repair (uvrC) and SOS (umuC).

Strains with mutations in endA, the gene encoding DNA-specific endonuclease I, exhibit reduced degradation of plasmid DNA, thus improving the yield and quality of DNA isolated from plasmid preparations. Because E. coli restricts (degrades) methylated DNA (most eukaryotic genomic or cDNA), it is imperative to use a strain in which these restriction systems (hsdSMR, mcrA, mcrBC, and mrr) are disabled when working with such nucleic acids. But if investigators want to generate unmethylated DNA, in order to use methylation-sensitive restriction enzymes, for instance, strains that lack dam or dcm activity can be useful.

Other markers enable or disable bacteriophage infection. The tonA mutation, for instance, confers resistance to T1 and T5 bacteriophage and subsequent cell lysis. But strains harboring the F' plasmid, which enables infection with the M13 virus, are useful for generating single-stranded DNA or phage display libraries.

PROTEIN EXPRESSION

Courtesy of Doug Lundberg

In the glare of a scanning electron microscope, the tiny E. coli looks like a rod. Each cell measures about 2 μm × 1 μm.

Researchers who want to express recombinant proteins in competent cells should consider strains that are specially designed to overcome difficulties such as inactive or insoluble proteins, codon bias, and toxicity. These strains often include an inducible T7 promoter-based system, to churn out genes under the control of that genetic element. The BL21 strain is widely used because it naturally lacks certain proteases, such as lon and ompT. Additionally, a strain that is RNase-deficient is useful to prevent mRNA degradation.

PACKAGING FORMAT

Packaging convenience is another important consideration. Most commercially available, chemically competent cells are packaged as 0.1 ml or 0.2 ml aliquots, which is enough for two to four transformations. Many companies now offer single-use 50 μl aliquots, which reduce waste and preserve transformation efficiency by eliminating freeze-thaw cycles. Also, 96-well plates containing predispensed competent cells are popular for high-throughput applications.

SPECIALIZED BUGS

Large manufacturers of competent cells carry even more specialized strains. Novagen, for example, offers the Veggie™ brand of competent cell lines, which are manufactured with non-animal-derived media and reagents. These strains are designed for downstream applications that require animal-free conditions. Both Stratagene and Novagen offer strains designed specifically for expression of proteins containing rare codons, while Invitrogen's product line includes cells designed to take up large inserts.

Hillary Sussman hillary@sciwriter.com is a freelance writer in Port Jefferson, NY. Aileen Constans can be contacted at aconstans@the-scientist.com.