Defining the steps in adaptation to lactose

ß-galactosidase activity appears in E. coli cells only after the addition of lactose.

So the question arises, does lactose activate pre-existing ß-galactosidase or does it induce its de novo (new) synthesis?

Melvin Cohn, working with Monod, searched for compounds that would mimic (agonists) or inhibit (antagonists) the effects of lactose.

A particularly useful class of compounds are the chromogenic substrates. ONPG and X-GAL are both colorless in solution.

When cleaved by ß-galactosidase ONPG produces an intensely yellow compound while X-GAL produces an intensely blue one.

It is possible to measure ß-galactosidase activity by measuring the appearance of the colored product

It is worth remembering that the ONPG and X-GAL assays measure enzyme activity, not the total amount of ß-galactosidase protein present.

Typically, to measure protein levels we would use other (typically antibody-based) method.

Cohn and Monod, produced antibodies specific for ß-galactosidase. This enabled them to follow both enzyme activity and protein levels.

They found that the appearance of ß-galactosidase activity correlated with the appearance of ß-galactosidase protein.

In order to understand how E. coli adapt to the presence of lactose, we will perform a genetic analysis.

We will search for mutant cells that either fail to grow on lactose as their sole carbon/energy source or which express ß-galactosidase all of the time, i.e. constitutively.

revised 9 July 2003