As previously mentioned, β-galactosidase cleaves lactose into galactose and glucose. However, these products of the natural reaction are not easily quantifiable. That is why instead of lactose, we introduce ONPG to the cells. (ONPG, o-nitrophenyl-B-galactoside, is a substrate but not an inducer of the lac operon.) ONPG, colorless, is cleaved by β-galactosidase into galactose and orthonitrophenol, which is yellow, has a λmax of 420. The amount of o-nitrophenol can be quantitatively reported by looking at the change in the absorbance at 420 nm. The ΔA420/min is the total rate of the enzyme’s activity. To find the specific activity of β-galactosidase in relation to cell concentration, the rate is divided by the concentration of bacteria cells. Thus, the specific activity can be expressed by the following equation: ΔA420/min/OD600/ml. If the culture is not in log phase and in the death phase, dead cells contribute to the optical density but they can’t produce β-galactosidase to contribute to the total activity, leading to a deflated specific activity value for the enzyme.
Let's go it over step by step, shall we? I'll walk you through it.
A culture in the log phase is needed, as inducing gene expression works best during this phase. Inoculate liquid broth with E. coli and grow it at 37⁰ C. The broth shouldn’t have any glucose, as this would block any lacZ expression. Using the broth as a blank, transfer the culture into cuvettes and take the OD600 of the culture until it reads approximately .5-.6. In a tube with the culture, add the phosphate Z buffer, which keeps β-galactosidase working efficiently. Add a drop of sodium docecyl sulfate (SDS) and a drop of chloroform, which will lyse the cells, stop translation, but leave β-galactosidase intact and available for ONPG hydrolysis. Keep this in a room temperature water bath until the temperature has equilibrated. It should be stable for a while, preventing time errors. Add ONPG to each tube being assayed. This will be time 0, the start of the reaction. An Eppendorf tube with distilled water, Z buffer, and ONPG should be used as a control to make sure hydrolysis of ONPG is because of the enzyme, and not a spontaneous reaction in the mixture. If, after 5 minutes, the tubes turn yellow too quickly, the bacterial culture should be diluted and re-assayed, because that small window of time means small time errors will skew the results significantly. In addition, there needs to be an excess of ONPG during the assay, to ensure that the rate of hydrolysis is limited by, and therefore determined by, the enzyme concentration. If not enough ONPG is available, the increase in A420 would not correlate with increases in expression of galactosidase. The start of induction for this enzyme takes about 15-30 seconds, with a logarithmic increase when plotted against time, so sometimes reactions can go to completion is inappropriate amounts of ONPG are used. Use sodium carbonate (Na2CO3) once the yellow color is observed to stop the reaction. Na2CO3 inhibits β-galactosidase activity, thus stopping ONPG hydrolysis. Take the tubes and centrifuge them for 10-15 minutes at 5,000 g. Transfer the supernatant to cuvettes and measure the OD420. Visually confirm the readings are reasonable. A420 should be higher the darker the yellow. If the pellet and supernatant are not separated properly, the cells will scatter the light and invalidate the readings. During the entire reaction period, the temperature must be kept constant, because variable temperatures will affect enzyme activity and absorbance values.
The change in A420 can be converted to moles of ONGP converted to ONP+ by using Beer’s law and plugging in the molar extinction coefficient for ONP+, 4500/M/cm. The moles of ONP+ converted can be divided by the turnover rate of β-galactosidase to find out how much β-galactosidase is active.
The assay is used to determine relative expression of the lacZ gene and the specific activity of β-galactosidase, the lacZ gene product, in a bacterial culture. In uninduced cultures vs. fully induced cultures, there is a 1000% difference in β-galactosidase activity. Using this assay, the amount of time that it takes for the lac operon to become fully induced in a culture and be determined. Most importantly, we can manipulate the operon and insert a set of other structural genes downstream of the lac promoter by homologous recombination. Presence of a neoR cassette, too, allows us to select for these transgenic cells. Since the sequence will under the promoter’s regulation, the expression of the genes inserted is proportional to the expression of lacZ. Thus, we can indirectly determine expression of the inserted sequence by assaying B-galactosidase activity.