Screening the mutants

You can prepare a variety of mutant organisms by exposing a culture to uv irradiation, then plating a dilute suspension onto an agar gel containing medium supplemented with coenzyme X. This will allow all the viable organisms to grow.

These colonies can be blotted onto a fresh agar gel, containing minimal culture medium - i.e. not supplemented with coenzyme X. Comparison of the two plates will allow you to see:

• which colonies have active coenzyme X synthetase - they grow as well as on the supplemented medium
• which colonies have impaired activity of coenzyme X synthetase - they form smaller colnies than on the supplemented medium
• which colonies have no activity of coenzyme X synthetase - they do not grow at all (and are shown in the example below by open circles to mark where there are viable colonies on the plate containing supplemented medium)

You select a colony to study by clicking on it on the left-hand (supplemented medium) plate; this allows you to investigate the change in the exon of interest in each mutant strain of Spuriosum hypotheticum.

By comparing the amino acidsequence in the wild-type and the mutant strains, and the effect this has on viability on minimal medium, you can begin to draw conclusions about the active site of the enzyme.