Searching for mutations in lactose utilization

This makes it relatively straightforward to identify mutations in the lactose utilization pathway.

In any population, the number of organisms that carry the specific mutations we are looking for is liking to be quite small, perhaps one in a million or less.

How do we find these 'needles in a haystack?'

There are two general ways to search for mutations that produce a specific phenotype.

You can select for or screen for the phenotype.

In a selection for a phenotype , you search for those organisms that grow under a specific set of conditions; normal "wild type" organism do not grow under these conditions, so only the mutants will appear.

For example, bacteria that carry mutations that make them resistant to bacteriophage (virus) infection can grow in the presence of phage, whereas wild type bacteria cannot.

It is also possible to select against a phenotype . Here you look for mutants that cannot grow under conditions where the "wild type" organism does grow. Here it is necessary to define permissive and conditional environments for the phenotype in question (see below).

In a screen you look to identify those organisms that display the phenotype you desire.

We will use a screen to identify mutant E. coli that cannot grow on lactose or that express ß-galactosidase activity all of the time.

Screening for conditional mutations

To isolate mutations that specifically block E. coli's ability to grow on lactose, we establish two distinct environments:

in the permissive environment mutant cells can grow;
in the restrictive environment they cannot.

We "ask" each bacterium, can you grow on lactose?

We will call those that cannot "lactose-minus" or lac- cells.

This "asking" is done using a classic method known as replica plating.

Most of the colonies on the glucose plate also grow into colonies on the lactose plate - they are lac+.

If we are lucky, however, a few colonies will fail to grow on lactose. These are, by definition, lac-.

revised 9 July 2003