Beyond the Lac-Operon: Part One   Carl W. Vermeulen, PhD Science-Projects.Com Williamsburg, Virginia science_projects@yahoo.com

Beyond the Lac-Operon: Part One

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We know all about the lac-operon. Or do we? Recently a girl in London asked her teacher: "But that cannot be all there is to the control of lactase activity. The lac-operon is only an on/off switch. There must be some sort of speed control." She and a half dozen others have worked together through "Dr. V's" website and have since presented their work to the scientific community at three different annual meetings. They have shown that competitive feedback control operates directly on the lactase molecules to govern their reaction rates. While what they found was not unexpected, their work forms the basis of a very neat sequence of lab exercises that take students through the lac-operon and beyond to the enzymological aspects of both qualitative and quantitative control of lactase's activity. These exercises are fully compatible with labs in genetics, microbiology and biochemistry - and certainly for A.P. Biology (labs 2, 6, 7 and some of 5) - all using the same system and much the same supplies (cutting costs). What is more, the enzyme inhibitor is not toxic such as are the usual mercury and lead based inhibitors formerly used. Here the inhibitor is a common sugar.

The VAST workshop was aimed at acquainting teachers with the bacteriological aspects needed to run this and the sequel (Part Two). This Part One shows how the in vitro enzymology of store-bought lactase can be extended to in vivo situation. The teachers find that true aseptic techniques may be put aside under the right conditions by using E. coli and MacConkey agar.

Warning! Know your suffixes: -ase = enzyme; -ose = sugar.

In vitro: Qualitatively various sugars were tested for inhibitory effects on lactase activity. Participants are introduced to the word "analog" and that ONPG is a structural analog of lactose. When lactase snips ONPG, the solution becomes yellow. The question is: when lactase finds itself in the presence of other sugars, can it snip ONPG. Using the setup shown to the right, 3 drops of ONPG were added to each tube which were watched to see which ones turned yellow. Inhibitory sugars prevented yellow formation.

In vivo: Qualitatively, the inhibitory sugar found will be added to a petri plate containing lactose, and then a mutant strain of E.coli that is unable to digest that sugar (but is able to digest lactose) will be grown on that plate. If the lactose is digested, the bacteria grow as deep red colonies; but if inhibited, they grow as pale pink.

An important pedagogical point is that while exhibiting something interesting in vitro, it would be purely academic unless it is found also to occur in the living system.