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Transformation and
IX.19 competent cells
Preparation and transformation of competent E. coli cells
Most nucleic acid fragments cannot enter bacteria under their own power. They need assistance traversing
the outer and inner cell membranes and reaching the intracellular site where they can be expressed and
replicated. The methods to achieve these goals fall into two classes:
- chemical and
- physical (Sambrook and Russel 2001).
Chemical methods, salt-competent cells
One of the breakthroughs in genetic engineering was the observation of Mandel and Higa in 1970 that E. coli
cells that had been soaked in an ice-cold salt solution can uptake purified  phage DNA. Traditionally, a
solution of 50 mM calcium chloride is enough to yield 105-106 transformed colonies of E. coli per µg plasmid
DNA. More effective variations of this basic technique use complex cocktails of divalent cations, notably
rubidium chloride. The reason why this procedure works remains still obscure. Possibly calcium chloride and
other salts cause the DNA to precipitate onto the outside of the cells, or perhaps the salt is responsible for
some kind of change in the cell wall that improves DNA binding. Generally, salt treatment affects only DNA
binding and not the actual uptake into the cells.
The actual movement of DNA molecules into salt-competent cells is stimulated by a temperature shock
(short incubation at 42 °C) followed by a short cooling step on ice. As it is said for preparation, the exact
reason why heat-shock is effective in the DNA uptake is not understood (Figure 1).
Physical methods, electro-competent cells
Exposure of cells to an electric charge destabilises cell membranes. It induces the formation of transient
membrane pores through which DNA molecules can pass.. This method was originally developed to
introduce DNA into eukaryotic cells and is now used as electroporation to develop electro-competent E. coli
cells for transformation experiments. It is the easiest, fastest, most efficient, and most reproducible method
for transformation of bacterial cells with DNA. With electroporation transformation efficiencies of more than
1010 transformants/µg DNA have been achieved and vectors in the size from 2.6 to 85 kb can be introduced
in electro-competent cells.
The DNA may be introduced into bacteria simply by exposing them to a short high-voltage electrical
discharge (Figure 2), (Sambrook and Russel 2001, changed).
Figure 1. Preparation and transformation of saltcompetent cells (Brown 1991)
Figure 2. Electroporation
A sharp short pulse of electricity
causes dimpling of membranes
followed by formation of transient
hydrophobic pores. While the pores
are open, DNA molecules can
easily pass from medium into the
cytoplasm. Reclosing of pores
seems to be a stochastic process
that can be delayed by keeping the
cells at low temperature (Bio-Rad
Lab.).
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