What is Bacterial Transformation?
Bacterial transformation is a process whereby foreign or exogenous DNA is introduced into a bacterial cell.
Although this phenomenon also happens in nature, bacterial transformation typically refers to a lab procedure whereby bacteria are forced to take up plasmids for a variety of reasons, such as: to propagate already constructed vectors i.e. to obtain more of a certain plasmid for a downstream application, or during cloning workflows, e.g., to isolate plasmids that have been recombined with specific genes or DNA sequences for cDNA library synthesis, recombinant protein production or transgene expression.
How Are Bacteria Transformed?
During transformation, plasmids are mixed with competent bacterial cells in the presence of a transformation buffer containing chemicals that enhance DNA uptake. The mixture is typically then either heat-treated or subjected to brief electrical pulses before spreading out on agar plates containing an antibiotic, e.g., ampicillin or kanamycin.
Transformed bacteria are selected for their ability to multiply and form colonies in the presence of the antibiotic. Expression of an antibiotic resistance gene present on the plasmid allows only the transformed bacteria to degrade the antibiotic, thus allowing them to propagate while untransformed cells die off.
What Exactly Are Competent Bacteria?
Lab strains of E. coli are most widely used in transformation experiments. Since E. coli strains are not naturally competent, they must be pre-treated in order to take up foreign DNA. There are multiple approaches to preparing competent cells, and these usually involve treatment of cells in the logarithmic growth phase with chemicals (chemically competent), heat shock (heat shock-competent), electrical impulses (electro-competent), or combinations of all three.
These treatments essentially create pores in the bacterial cell membrane that are big enough for plasmids to pass through but without destroying the membrane. Competent cells are then usually snap-frozen in liquid nitrogen and then stored at -80 °C until use.
Factors Affecting Transformation Success
There are many transformation protocols available, and most labs have their own variations on these. The procedure itself is often simple but the success depends on a number of factors. The main prerequisite is high-quality competent cells.
Other protocol-dependent factors that impact success include handling time, culture media, plasmid DNA concentration, volume and purity, whether or not the plasmid is intact or linearised (i.e. when ligation reactions are used as the transforming material), as well as reagents present from previous reactions. For example, when transforming with ligation mixtures, the DNA ligase must be deactivated as it may interfere with transformation, e.g., it inhibits electroporation.
Back to Those Competent Cells Again!
Many labs have their own protocols for preparing competent cells, but for certain applications that require high transformation efficiency, consistent results and/or a high throughput workflow with time-saving steps, it may be worthwhile investing in pre-made competent cells that have gone through rigorous quality control processes.
There are a number of competent cell products on the market, and some of these are prepared in specialised buffers that bypass the need for any incubation steps with transforming DNA, to greatly speed up your workflow without compromising on success!
Get Started With Nordic BioSite!
Nordic BioSite has a broad range of competent cells as well as transformation kits that combine competent cell preparation with transformation, and these span a range of popular E. coli lab strains, including DH5a, JM101 and its derivative TG1, HB101, XJa and XJb, and others. If you would like to know more about our portfolio or ask questions about any aspect of transformation, please get in touch with us at email@example.com