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Worksheet 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.). page: