by Jeffrey Graf

Created on: March 22, 2010

The bacterium  E. coli is the workhorse of the molecular biology lab. The biotechnology industry has grown up around using this common bacterium, an intestinal bacterium common to warm blooded animals, to multiply or expand a sequence or segment of DNA for future biotechnological use. The segment usually represents a gene, and we usually refer to this as cloning a gene.  The characteristic which makes E. coli such a valuable partner is it's propensity to engulf, consume, or otherwise endocytose, free fragments, or loops of DNA known as plasmids. This property has been further enhanced in proprietary strains of E. coli which are in fact are the only ones that are actually useful in the laboratory.  Where as eukaryotic cells, such as mammalian cells, must be transfected using viral vectors or other complex means, a little cesium salt on loop DNA is all laboratory strains of E. coli need to make a meal.

In practice though technicians need to know which cells have transfected a desired plasmid and which cells have not. The common way is to place an antibiotic resistance gene on the plasmid, and put a small amount of antibiotic ( ampicillin )  in the culture media. By doing this, only cells that have taken up plasmids will create colonies will survive.

We are not done. Of those cells which have taken up plasmids, we need to know which cells have plasmids which have our desired insert, or gene that we want to clone. To observe this, we need to make a color marker for each of the colonies that are formed on our plating dish. The reaction that we take advantage of is that if a chemical called X-gal is placed in the culture medium, and the colonies of interest are producing an enzyme called beta - galactosidase, then X-gal is digested into two products, a sugar known as galactose and a blue marker. Thus colonies that are expressing an active beta- galactosidase turn blue.

Researchers have found that beta - galactosidase can be disabled by deleting a portion on either end of the gene.[2]  The gene can be rescued by expressing that portion of the gene on a different segment of DNA, or plasmid. The two fragments of the beta - galactosidase gene are then referred to as the alpha fragment and the omega fragment. When they are expressed sucessfully together, it is called alpha - complementation. In practice, one fragment is expressed on the plasmid, and one, complementary fragment, is expressed in the proprietary E. coli cell line

• 1
• 2
• Next Page >>