All organisms (humans included) work hard to use their resources efficiently. For example, if you lived in the warm and sunny Florida, you wouldn't spend your hard earned cash on snow chains for tires. But here in the frigid and blizzard-ridden northeast, snow chains might be a very sensible investment during the winter blizzards. However, during the summer, you would pack those chains away, even in here in NY!

Microorganisms also strive to spend their resources wisely. In any point in time, they only make the enzymes they need to live and grow at that moment. How do they "know" which enzymes they need? It all depends on what's in their environment at the time.

For example, a sugar like lactose is not very common in the environment, so bacteria only make the enzymes needed to use lactose when lactose is around. The presence of the lactose molecule actually causes the bacterium to begin transcribing and translating the lactose genes-- that is, it induces lactose the synthesis of enzymes that break down lactose.
However, glucose is a very common, and much preferred, carbon and energy source. When glucose is available, the synthesis of all other sugar-using enzymes is stopped (through a process called "catabolite repression"). Glucose shuts down the transcription and translation of all other sugar genes-- that is, glucose represses the production of other sugar-utilizing enzymes.

Of course, we can't really "see" what is happening around a bacterial chromosome, to tell if genes are being "turned on" or "turned off". But we can do experiments that give us the same information. One such experiment is to measure the activity of the enzyme in question under certain growth conditions. By doing this, we can learn when the enzyme is being made, and when it is not!

Measuring Enzyme Activity

Escherichia coli by measuring enzyme activity.

Protocol

In this experiment, you will be causing the induction and catabolite repression of the lac operon under a variety of different conditions. Because we cannot see if the lac operon is turned on or off, we must invent cleaver ways to observe whether or not the lac operon has been transcribed.

One such clever technique is to use o-nitrophenylgalactoside (ONPG). This molecule is composed of galactose bonded to nitrophenol. B-galactosidase will recognize this bond as a substrate, and cleave this molecule to produce galactose and o-nitrophenol.


The ONPG and its cleavage products (Galactose and o-Nitrophenol) are colorless. The o-nitrophenolate which results from the pH increase to greater than 8, appears yellow. The absorption of o-Nitrophenolate can be measured at 420 nm.

If ONPG is available in excess, then the amount of o-nitrophenolate produced is proportional to the amount of enzyme (B-galactosidase) present. The reaction is stopped by increasing the pH to 11(with Na2CO3), which inactivates B-galactosidase.













The first part of the experiment involves setting up the cultures and allowing time for induction (if any) to occur. (see diagram below). As with every experiment, you must have the appropriate controls, to prove that the experiment is working properly.

The second part involves the enzyme assay with ONPG to measure the amount (if any) of B-galactosidase that was produced. After 1 hour at 37¡C, each tube is assayed for B-galactosidase activity by first lysing the cells (by adding SDS and chloroform), then by adding ONPG. The reaction is stopped by adding Na2CO3.

Why didn't the tube with both glucose and lactose turn yellow?