Beta Galactosidase And Glucose Lab Report

This experiment was conducted in order to demonstrate and understand the control of the lac operon in the presence and absence of lactose and glucose. The lac operon consists of a promotor, operator, and three structural genes. In the promoter, DNA sequence regulates when DNA goes off and on. It is a binding site for RNA polymerase. RNA polymerase recognizes the sequence in the promoter, finds the beginning, and starts to transcribe. In the operator, however, as RNA polymerase begins to transcribe, it is stopped by a protein called the repressor which binds and sits on the operator. With no glucose and with the presence of lactose, the lactose will bind to the repressor changing its shape which will lead to its dissociation. This will allow

The three genes are lac Z, lac Y, and lac A encode the enzymes beta-galactosidase, permease, and transacetylase respectively. In this experiment, we will measure the enzyme activity of beta-galactosidase which was induced when lactose was added to a 50ml log phase E. coli culture grown with glycerol. Another E. coli mixture grown with glycerol and glucose was also used. Eight tubes were labeled with 0, 5, 10, 15, 20, 25, 30, and G-30. Toluene was added to each tube in order to make holes in the E. coli cell membrane which killed all of the bacteria preventing any further changes in beta-galactosidase levels. 2.5 ml of 5% lactose was added to the E. coli mixture grown in glycerol only. Once lactose was added, 2ml of the mixture was added to the tube labeled with 0 min. The remaining E. coli mixture was put in a 37°C shaking water bath and a timer was started. After 5 minutes, the mixture was taken out of the shaker and added to the test tube labeled with 5 min. The same was done for the other test tubes except for G-30

ONPG produces the yellow color. The time was recorded when each tube turned yellow and incubated for 5 minutes in the water bath. 1ml of sodium carbonate (Na2CO3) is added to each to tube in order to develop the color as well as stop the reaction between ONPG and beta-galactosidase which will in turn stop the enzymes activity allowing us to measure the absorbance with no rush. The tube that turns yellow first is the one that has the highest levels of beta-galactosidase. It was hypothesized that the tube with the greatest amount of beta-galactosidase is produced in the tube that had the lactose mixture in the shaker for the longest time. In this case, the tube that contained the E. coli mixture with glycerol and lactose for 30 min turned yellow first. The absorbance was measured for each tube at 420 nm and at 550 nm. Using the absorbance, the enzyme unit per cell is calculate using the following equation where OD is the absorbance and t is the time of incubation with ONPG plus the 5 minutes of incubation in water