What function of magnesium chloride in making competent cells?

Answer 1

Since E. coli is not naturally transformable, the ability to take up DNA or competency must be induced by chemical methods using divalent and multivalent cations (calcium, magnesium, manganese, rubidium, or hexamine cobalt) . Alteration in the permeability of the membranes allows DNA to cross the cell envelope of E. coli which is composed of an outer membrane, an inner membrane, and a cell wall. The outer membrane of E. coli can be understood by application of the fluid mosaic model for membranes and is composed of phospholipids, proteins, and lipopolysaccharides. Many channels or zones of adhesions are formed by the fusion of the outer membrane and the inner membrane through the cell wall layer. Although the transformation mechanism is not known, previous studies indicate that these channels allow for the transport of DNA molecules across the cell membrane. The negative charges of the incoming DNA, however, are repelled by the negatively charged portions of the macromolecules on the bacterium's outer surface. The addition of CaCl2 serves to neutralize the unfavorable interactions between the DNA and the polyanions of the outer layer. The DNA and competent cells are further incubated on ice for thirty minutes to stabilize the lipid membrane and allow for increased interactions between calcium ions and the negative components of the cell. The reaction mixture is then exposed to a brief period of heat-shock at 42oC. The change in temperature alters the fluidity of the semi-crystalline membrane state achieved at 0oC thus allowing the DNA molecule to enter the cell through the zone of adhesion .

Prior studies showed that some E. coli strains are more susceptible to transformation than others due to differences in the composition of the lipopolysaccharide. E. coli with a lengthy O-linked polysaccharide blocks or hinders DNA from entering the cell. Adding magnesium to the media increases transformation yield by enhancing the ionic interaction of the molecules on the surface and therefore alters the suppleness of the membrane for more efficient transformation.

Answer 2

Magnesium chloride stabilizes the cell membrane and promotes DNA uptake in the transformation process when making competent cells. It helps neutralize the negative charges on the DNA phosphate backbone, allowing for more efficient binding to the cell membrane and uptake into the cell.