Before the experiment began, students were prompted to create procedures that met the goals of the experiment. These goals were to develop an experimental understanding of enzyme kinetics and determine the kinetic parameters for the β-Galactosidase enzyme. To increase the rate of the experiment, a collaborative setting was established. The class was split into two collaborative teams, with each team further split into three separate groups to divvy up responsibilities. Upon the completion of brainstorming, a procedural strategy to meet the goals of the lab was formed.
For the first part of this experiment, the affects of changing the concentration of β-Galactosidase on the rate and product formation of ONP were investigated. In order to execute this investigation, a Z buffer solution was prepared in order to create an optimal environment for β-Galactosidase activity. A 50 mL working solution was prepared by the combination of 50mM potassium phosphate, 40mM potassium chloride, 1mM of magnesium sulfate, and deionized water. This stock solution was prepared to be 10x concentrated, allowing for the subsequent solutions to be diluted down to their respective molarities. Each group created one of the stock solutions. To…show more content… A β-Galactosidase enzyme stock concentration of 5 mM was prepared by dissolving 100 mg of the enzyme into 20 mL of the stock buffer solution. From the stock enzyme solution, each group serially diluted three different enzyme concentrations for a total of nine different enzyme concentrations to be investigated: 2.25 mM, 2.0 mM, 1.75 mM, 1.5 mM, 1.25 mM, 1.0 mM, 0.75 mM, 0.5 mM, 0.25 mM. The substrate concentration was kept constant while experimenting on the affect of varying enzyme concentration on the rate and product formation of ONP. An ONPG substrate stock concentration of 2.5 mM was prepared by dissolving 50 mg of substrate into 20 mL of the stock buffer
