This experiment is designed to show that bacterial cells possess control mechanisms which regulate cell metabolism. This phenomenon, in the case under consideration, results from the preferential biosynthesis of the enzyme, beta-galactosidase. You are expected to be resourceful given the briefness of this description of the lab protocol. Read and recall any available information about beta-galactosidase induction. Confer first with your lab partner. If there are questions about further aspects of technique ask other groups and your lab instructor.

For this experiment an exponentially growing culture of E. coli wild type (grown on a minimal glucose medium) will be provided. Also, sterile flasks containing medium (100 m1) supplemented with (1) glucose (2) lactose or (3) glucose plus lactose will be provided. Although the flasks are initially sterile, we will depend on the overwhelming number of E.coli bacteria that you are innoculating the flasks with to allow you to ignore contamination of you cultures during the course of the lab.

Each student is to inoculate one flask of each type of medium (to about 0.1 OD units at 540nM in a spectrophotometer) and grow the culture at 37°C with shaking. OD readings of the cultures of (1) glucose and (2) lactose will be determined at 30 minute intervals. The turbidity of the culture which contains glucose plus lactose is to be measured at 15 minute intervals and a 1.5 ml aliquot of this culture is collected at the time of each reading in order to assay the lactose-utilizing enzyme, B-galactosidase. Plot the log of the OD reading vs. time for each culture (use the same graph for all three cultures).

beta-galactosidase assay

For this enzyme assay, 1.5 ml aliquots of culture are pipetted into small test tubes which contain 1 drop of toluene. The tubes are shaken vigorously and then incubated for 30 minutes at 37°C. The tubes are then placed in the deep freeze until the next lab period.

At the start of the second lab period the tubes are thawed and placed in a 28° water bath. Then quickly 0.2 ml of a solution of M/75 o-nitrophenyl-B-galactoside in M/4 Na phosphate, pH 7.0 is added. The tubes are to be quickly shaken and then incubated at 28C for 15 minutes. The reaction is then terminated by addition of 0.5 ml of 1 M Na2CO3, and the optical density is measured at 420 mu in the Spectrophotometer

Analysis

One mu mole/m1 o-nitrophenol has an optical density of 0.0075 under the above conditions (using 10 mm light path). Calculate the specific activity (i. e. u moles o-nitrophenol produced/ mg protein /min) of the most active sample (OD units/cell number should be calculated assuming the turbidity of a serial dilution of an exponential overnight culture to reach 10⁹ cells per ml.). Is there any missing information? Plot mu moles o-nitrophenol released vs. time on the same graph that you used for the growth curves. Is there a correlation between the growth curve of the glucose plus lactose culture and the amount of B-galactosidase found in the culture. Explain.