Recombinant DNA

The Bacterium E. coli plays a pivotal role in what is popularly known as genetic engineering. Our detailed knowledge of its microbiology, physiology, and biochemistry has allowed us to develop mutants that can be used to clone pieces of DNA from most known organisms, safe in the knowledge that the strains harboring foreign DNA (recombinants) cannot survive outside the laboratory. Adapted E. coli plasmids are used to maintain foreign DNA in an easily accessible form (plasmids known as cloning vectors). Expression vectors are plasmids used to generate large amounts of recombinant protein (sometimes up to 10% of the total cell protein) so that enzymes can be purified for research, biotechnological, food, and medical uses.  Shuttle vectors can also be constructed, which allow the easy creation of specific arrangements of genes or gene fragments in E. coli before the construct is transferred to another organism via transformation, transduction, electroporation or transfection (the  latter two techniques are not  discussed in this text). As the demand for very specific proteins subject to post-translational modification becomes more sophisticated, other organisms have  been used for cloning and  expression, but E. coli is still the most widely used.

Part of the attraction of E. coli is the ease with which it’s genomic and plasmid DNA can be isolated. E. coli, in common with many other bacteria, will lyse on the addition of a sodium hydroxide solution. After neutralization, the genomic DNA can be purified by techniques such as cesium chloride density centrifugation or by the use of the many commercially available spin columns. The many plasmids used in biotechnology can also be separated from the rest of the cytoplasmic contents after alkaline lysis. If the lysate is treated with sodium dodecyl sulfate (SDS) and neutralized with sodium acetate, larger macromolecules (proteins, membrane debris, and genomic DNA) become entangled (both physically and chemically) with the white precipitate formed by SDS. The plasmids remain in solution and can be purified further with a few simple steps. This means that plasmid DNA can be isolated from E. coli in less than 15 minutes, rendering the organism suitable for building up libraries of DNA fragments in plasmids for screening projects or archiving.