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How are competent bacterial cells transformed with a plasmid?

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There are two primary methods for transforming bacterial cells: heat shock and electroporation. In both cases, the bacterial cells have to be made competent or permeable to plasmids that you would like the cell to propagate. To create competent cells for either transformation method used, bacterial cells are grown to logarithmic phase and harvested. Cells growing exponentially can be rendered competent more easily than cells at other stages of growth. After harvesting, the cells are treated differently. Chemically competent cells are created using a series of cold salt washes to disrupt the cell membranes, preparing the cells to accept plasmid DNA(1) (2) . For electrocompetent cells, the cells are chilled and washed with cold deionized water and 10% glycerol(3) . A low-salt environment is important when electrical currents are involved.

To introduce the desired plasmid into chemically competent cells, the plasmid DNA is mixed with chilled cells and incubated on ice to allow the plasmid to come into close contact with the cells. The plasmid-cell mixture then is briefly heated to 45–50°C, allowing the DNA to enter the cell through the disrupted membrane. The heated mixture is then placed back on ice to retain the plasmids inside the bacteria. Many cells do not survive the rapid temperature change but enough maintain integrity to keep the plasmid and, when medium is added, recover and divide.

For electroporation, the competent cells also sit on ice with the plasmid DNA. However, the plasmid-cell mixture is exposed to an electrical current, opening pores in the cell membrane so that the plasmid can enter the cell. Some cells do not survive this treatment but many are able to replicate once medium is added. If the plasmid DNA solution has too much salt in it, arcing can occur, compromising the transformation.

Depending on the transformation method used, a plasmid can enter the cell through holes or pores in the bacterial cell wall created by salt washes and heat treatment or no-salt washes and electroporation. Both methods allow efficient recovery of transformed cells using antibiotic selection for the plasmid of interest.

References

  1. Hanahan, D. (1983) Studies on transformation of Escherichia coli with plasmids J. Mol. Biol. 166, 557–77.
  2. Hanahan, D. (1985) In: DNA Cloning, a Practical Approach. Volume I. Glover, D.M. ed. IRL Press, Ltd. London, UK. 109.
  3. Seidman, C.E. (1994) In: Current Protocols in Molecular Biology, Ausubel, F.M. et al. eds., John Wiley and Sons, NY.

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