b-galactosidase is an enzyme which hydrolyzes b-D-galactosides. After addition of the gratuitous inducer, isopropylthiogalactoside (IPTG; a non-metabolizable analog of lactose) to growing E.coli, active b-galactosidase molecules are synthesized after a lag of 1.5 - 2.0 min. During this time, the repressor-operator complex dissociates and a specific mRNA is transcribed and translated into b-galactosidase monomers, which are then assembled into active oligomers.

This protocol describes a simple assay for induced b-galactosidase in situ that is easily done in class; it is a powerful way to illustrate regulation of gene expression. Once the enzyme has been induced (e.g. by IPTG), it is detected by assaying the enzymatic conversion of substrate (lactose or a derivative of lactose) into a (detectable) product. In this case, it can easily be measured with chromogenic substrates: i.e. colorless substrates which are hydrolyzed to yield colored products. An example is o-nitrophenyl-b-D-galactoside. This compound is colorless, but in the presence of b-galactosidase it is converted to galactose and o-nitrophenol. The o--nitrophenol is yellow and can be measured by its absorption at 420 nm. If the o-nitrophenol-b-D-galactoside (ONPG) concentration is high enough, the amount of o--nitrophenol produced is proportional to the amount of enzyme present and to the time the enzyme reacts with the ONPG. In order for the assay to be linear, the ONPG must be in excess. For best results, the amount of enzyme should be such that it takes between 15 minutes and 6 hours for a faint yellow color to develop. The reaction is stopped by adding a concentrated Na2CO3 solution, which shifts the pH to 11. At this pH, b-galactosidase is inactive.

An optional extension to this is to use the assay to actually study the process of induction: the experiment is designed to follow the b-galactosidase synthesized by a growing culture after the addition of IPTG. One purpose of this experiment is to use the data to determine the kinetics of "z-mRNA" synthesis during induction. We will see that the induction of mRNA starts without any measurable lag; or in other words, the maximal rate of transcription is instantaneously achieved. (Note: if you elect not to carry out this experiment, you may use the data obtained by us to make these calculations.)

2 drops SDS 4 drops chloroformb. Reaction1.) When the cultures reach the desired density, record the O.D.600. At that point, remove a 1.0 ml aliquot from each culture and start the timer (e.g. stopwatch). Add the removed aliquots to separate "t=0" reaction tubes; continue to incubate the remainder of the culture at 37oC with shaking. At t = 3 min., add IPTG to each culture. At t = 5, 15, and 30 min., remove 1.0 ml aliquots from each culture and add to the appropriately marked reaction tubes. 2.) After collecting the t = 30 min samples, measure the OD600 again (comparing this with the value obtained at the beginning of the experiment will tell you how much the culture has grown during the experiment; this information is important in making the calculations discussed later). 3.) To each reaction tube, add 0.4 ml ONPG and start your stopwatch. Incubate the tube at room temperature until you can see a yellow color develop OR 30 min.....whichever comes first. AT THAT POINT, ADD 1.0 ML OF THE NA2CO3 SOLUTION TO STOP THE REACTION; STOP YOUR STOPWATCH AND RECORD THE TIME. For a control, prepare a tube with twice the volume of reaction buffer, add ONPG, incubate and add Na2CO3 (this is to determine the extent to which ONPG hydrolyzes spontaneously). c. Assay1) For each tube, record the O.D.420; ideally, this (yellow color) reading should be 0.6-0.9. Record the OD550. The reading at 420nm is actually a combination of absorbance by the o-nitrophenol and light scattering by the cell debris. The latter component can be minimized (in the case of high levels of b-galactosidase) by assaying more highly diluted cultures or it can be virtually eliminated by transferring the reaction mixture to plastic microfuge tubes, centrifuging for several minutes, and carefully transferring the supernatant with a pasteur pipet to a new spectrophotometer cell. Alternatively, the light scattering can be corrected for by obtaining the absorbance at another wavelength (550 nm) at which there is only light scattering by debris (no absorbance contribution from o-nitrophenol). The light scattering at 420nm is proportional to that at 550nm: for E.coli, the OD420 light scattering = 1.75 x OD550. Using this correction factor, which compensates for the light scattering, the true absorbance of the o-nitrophenol can be computed. Enzyme units are calculated according to: The OD420 and OD550 are read from the reaction mixture. The OD600 reflects the cell density just before assay; t = time of the reaction in minutes, and v = the volume of the culture ( in ml) used in the assay. These units are proportional to the increase in o-nitrophenol per minute per bacterium. They are convenient because a fully induced culture grown on glucose has approximately 1000 units and an uninduced culture has approximately 1 unit. Sample calculation: OD600 of culture at time of starting reaction: 0.601 ml of culture was added to 1 ml reaction buffer plus ONPG, SDS, and chloroform; after 10 min, the reaction is stopped and the OD determinations were made:OD420=0.099OD550=0.05 2.) The specific activity of b-galactosidase is defined in terms of units/mg protein. Many investigators define a unit of b-galactosidase as the amount of enzyme which produces 1nmole o-nitrophenol/min at 28oC, pH 7. Under the above conditions, 1 nmole/ml o-nitrophenol has an OD420 = 0.0045 using a 10 mm light path. Therefore the above units can easily be converted into these units. Although protein is usually determined directly from extracts of estimated from the dry weight of cells, we can estimate the amount of protein by assuming that 109 cells yields approximately 150 mg protein, and that an OD600 of 2.0 corresponds to 109 cells/ml. Using this information, calculate the specific activity of b-galactosidase in terms of nmoles o-nitrophenol/min/mg protein. (Using these parameters, calculations in the literature indicate that 3 activity units shown above correspond to 1 specific activity unit )

1) When the culture reaches the desired density, record the O.D.600. At that point, remove a 1.0 ml aliquot from the culture and start the reaction timer (e.g. stopwatch). Add the removed aliquot to the "t=0" reaction tube; continue to incubate the remainder of the culture at 37oC with shaking.4 The remainder of the experiment involves removing additional 1 ml samples at t = 1,2,4,5,7,9,12,18,24,&32 with induction by addition of IPTG to the culture at t = 3.

All organisms need to be able to respond to changes in the environment around them and even bacteria are capable of regulating their metabolism depending upon conditions. When nutrients become available, a microorganism must be able to respond to and use them for growth, if the microbe is to compete and survive. A response to changes in the environment is often dependent upon the de novo production of enzymes to metabolize the nutrient in question. This is termed enzyme induction. The b-galactosidase operon was one of the first induction systems studied. A major study by Francois Jacob and Jacques Monad was seminal to our present understanding of bacterial gene regulation

The system investigated by Jacob and Monod involved the utilization of lactose by Escherichia coli. Lactose is a dimeric sugar and is acted on by the enzyme ß-galactosidase (b-gal), which adds water to the b-linkage, splitting it into glucose and galactose (Fig. 1). The products of this reaction are then metabolized via glycolysis. When lactose is not present in the environment, b-gal cannot be detected inside the cell. If lactose is added a 500-1000 fold induction of b-gal is observed. How does this induction take place?

Figure 1. The enzymatic degradation of lactose by b-gal.

To understand how b-gal is induced, it is first necessary to know the organization of the genes involved in the process. The lactose operon (Figure 2) is composed of 3 genes. b-galactosidase is coded for by the lacZ gene. The lacY gene codes for galactose permease, an enzyme responsible for the transport of lactose across the cellular membrane. The lacA gene codes for the production of the thiogalactoside transacetylase, whose purpose in lactose metabolism remains unknown. (When looking at almost any research problem there are always unanswered questions, such as lacA. That's what makes science fun!) The gene for the repressor of the lactose operon, lac I, is located upstream of the lacZYA genes.

Inthis experiment, the amount of b-gal present in the cell will be used to gauge the activity of the lactose operon and illustrate its regulation. How will b-gal activity be measured? We will use an enzyme assay.

An enzyme is a protein that catalyzes a biochemical reaction. Measurement of enzymes is accomplished by adding the compound the enzyme acts upon (called a substrate) to a reaction mixture containing everything necessary for the reaction to take place (protein, ATP, cofactors, etc.). The enzyme then acts on the substrate converting it to product. In one type of assay, (the kind we will be using) the increase in product or the decrease in substrate is monitored over a set time period. The final amount of product produced, or substrate lost, in a certain amount of time is proportional to the concentration of the enzyme and is often used to quantitate the amount of an enzyme present. This is termed an end-point assay. As an example, for b-gal, the substrate lactose, could be added to the enzyme in solution and the production of glucose or galactose could be measured.

Good enzyme assays have several features in common 1) absolute specificity, 2) high sensitivity, 3) accuracy and precision, 4) convenience. However, the in vivo substrates and products of the b-gal catalyzed reaction are all colorless. These products would be difficult to measure and this violates the convenience aspect of a good assay. Instead, an artificial substrate, ortho-nitrophenyl-b,D-galactoside (ONPG), is used (Figure 3). ONPG is a colorless compound that is split at its galactoside bond into galactose (colorless) and ortho-nitrophenol (ONP, which is an intense yellow color at high pH) by b-gal. The concentration of ONP can be measured using a spectrophotometer and related back to enzyme activity.

Enzyme activity is represented in units, which is defined as the number of micromoles of substrate converted to product in 1 min., usually at 30°C. The units of b-gal activity can be calculated using the concentration of ONP. See the end of this experiment for details.

In this experiment you will first learn how to perform one type of enzyme assay, an end-point assay and in the second part, the induction of b-gal activity in two E. coli strains will be followed under different conditions.

Today you will be practicing the b-gal assay. We will be performing this now so that next period, when time is limited, you will be old pros at the assay and it will not slow you down during the induction experiment.

In this experiment, each pair will test for the induction of b-gal under one of the following conditions:

These two strains have different phenotypes for lactose utilization. In the experiment, you will investigate these phenotypes. After analysis of the data and careful consideration of the genetics of the lac operon, it will be possible to hypothesize about the genotypes of the two strains. Each pair of students will be responsible for monitoring one flask. Divide the labor between the two of you (i.e. one person reads A600, the other performs the b-gal assay). The data generated in this experiment is most easily recorded in tabular form.

To calculate b-gal activity for this experiment gather together the A420 reading from the b-gal assay, the A600 turbidity reading, and the elapsed time of the assay. It is very useful to organize your data into a table. For example....

e = The extinction coefficient (1.1 x 103 ml/µmole•cm)

The goal is to compare the activity of b-gal in each strain. To derive b-gal activity, we next calculate the total amount of product in the tube, ending up with µmoles. To do this, multiply the total volume of the assay (in ml) by the concentration of ONP calculated in step 1.