Q.Why must the cell wall of a bacterial host be made 'competent' before it can take up foreign DNA? Describe briefly how CaCl2 treatment followed by heat shock helps bacteria take up plasmid DNA.
A bacterial cell wall is an effective barrier that normally prevents large, highly charged molecules such as DNA from simply diffusing into the cell. Without some intervention, mixing plasmid DNA with untreated bacteria would result in essentially no DNA uptake at all. This is why host cells must first be made 'competent' -- temporarily rendered permeable to DNA.
One widely used method treats the host bacteria with a specific concentration of calcium chloride (CaCl2), a divalent cation thought to destabilise the structure of the cell wall and outer membrane, making the cell more receptive to taking up external DNA. The CaCl2-treated cells are then mixed with the recombinant plasmid DNA and subjected to a carefully timed heat shock: the mixture is first kept on ice, then briefly transferred to a warmer temperature (commonly around 42 degrees Celsius) for a short, precise period, and then quickly returned to ice. This rapid temperature change is believed to create brief, transient pores in the cell envelope, or otherwise increase its fluidity for a short window, allowing plasmid DNA sitting close to the cell surface to be drawn inside before the cell wall reseals itself.
[!ANSWER] The combination of CaCl2 treatment (destabilising the cell wall) and heat shock (transiently increasing DNA permeability) is what allows plasmid DNA, which cannot normally cross an intact bacterial cell wall, to enter a competent host cell.
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