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
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.
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
Many researchers elect to purchase
Some DNA is particularly unstable, such as genes that exhibit secondary structure or contain long inverted repeats that may be deleted by
Strains with mutations in
Other markers enable or disable bacteriophage infection. The
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
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.
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.
Suppliers of Competent Cells
Active Motif (5)
BD Biosciences-Clontech (5)
Edge BioSystems (3)
Gene Therapy Systems (4)
Orion Biosolutions (2)
PGC Scientifics (6) (distributors for GeneChoice)
Zymo Research (2)