Extending the Scope of Direct Selection

Direct Selection would be very limited indeed if it could be used only for cloning antibiotic resistance genes. Fortunately the technique can be extended by making use of mutant straints of E. coli as the hosts for transformation.

As an example, consider an experiment to clone the gene trpA from Ecoli. This gene codes for the enzyme tryptophan synthase, which is involving in biosynthesis of the essential amino acid tryptophan.

A mutant strain of E.coli that has a non-functional trpA gene is called trpA-, and is able to survive only if tryptophan is added to the growth medium. E.coli trpA- is therefore another example of an auxotroph(as shown in image).

This E. coli mutant can be used to clone the correct version of the trpA gene, Total DNA is first purified from a normal (wild-type) strain of the bacterium.

Digestion with a restriction endonuclease, followed by ligation into a vector, produces numerous recombinant DNA molecules, one of which may, with luck, carry an intact copy of the trpA gene (a). This is of course the functional gene as it has been obtained from the wild-type strain.

The ligation mixture is now used to transform the auxotrophic E.coli trpA- cells. The vast majority of the resulting transformation will be auxotrophic, but a few now have the plasmid-borne copy of the correct trpA gene. These recombinants are non-auxotrophic - they no longer require tryptophan as the cloned gene is able to direct production of tryptophan synthase (c).

Direct Selection is therefore performed by plating transformants onto minimal medium, which lacks any added supplements, and in particular has no tryptophan (d). Auxotrophs cannot grow on minimal medium, so the only colonies to appear are recombinants that contain the clones trpA gene.

Next: The Scope and Limitation of Marker Rescue