Choosing the Right Bug

SELECTION STRATEGY:Courtesy of David McNeillThe genetically altered bacteria on this plate are easily detected under ultraviolet light. Escherichia coli were transformed with a plas-mid encoding the green fluorescent protein (GFP), which makes the colonies fluoresce under UV light. The transforming plasmid also encodes resistance to the antibiotic ampicillin, which allows the cells to grow on this antibiotic-containing agar dish. But in this image, the ampicillin resistance is leaking out of the

Hillary Sussman
Mar 28, 2004
<p>SELECTION STRATEGY:</p>

Courtesy of David McNeill

The genetically altered bacteria on this plate are easily detected under ultraviolet light. Escherichia coli were transformed with a plas-mid encoding the green fluorescent protein (GFP), which makes the colonies fluoresce under UV light. The transforming plasmid also encodes resistance to the antibiotic ampicillin, which allows the cells to grow on this antibiotic-containing agar dish. But in this image, the ampicillin resistance is leaking out of the transformed colonies, allowing some untransformed, nonfluorescent colonies to grow.

The true workhorses of molecular biology are neither fruit flies nor nematodes, neither budding yeast nor mice. No, the diminutive Escherichia coli has to get that accolade. These bacteria take up foreign DNA in the form of a plasmid or viral vector and make it their own. After thus transforming themselves, they churn out enough copies of the nucleic acid for scientists to collect the material for use...

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

<p>THE WORKHORSES OF MOLECULAR BIOLOGY:</p>

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.

Suppliers of Competent Cells

A complete listing of competent cells, including efficiency, genotype, packaging format, and price, is available on the Web at http://www.the-scientist.com. (Number indicates # of available strains.)

Active Motif (5) http://www.activemotif.com

BD Biosciences-Clontech (5) http://www.bdbiosciences.com

BioChain (2) http://www.biochain.com

Bioline (3) http://www.bioline.com

Bio-Rad (5) http://www.bio-rad.com

Edge BioSystems (3) http://www.edgebio.com

Epicentre (10) http://www.epicentre.com

Gene Therapy Systems (4) http://www.genetherapysystems.com

GrowCells.com (10) http://www.growcells.com

Invitrogen (31) http://www.invitrogen.com

InvivoGen (2) http://www.invivogen.com

Lucigen (5) http://www.lucigen.com

Novagen (51) http://www.novagen.com

Orion Biosolutions (2) http://www.orionbiosolutions.com

PGC Scientifics (6) (distributors for GeneChoice) http://www.pgcscientifics.comhttp://www.genechoiceinc.com

Promega (5) http://www.promega.com

Qbiogene (1) http://www.qbiogene.com

Sigma-Aldrich (9) http://www.sigmaaldrich.com

Stratagene (43) http://www.stratagene.com

Trenzyme (4) http://www.trenzyme.com

Zymo Research (2) http://www.zymoresearch.com

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