Lab C - Lab Manual Chapter_ 24W-LIFESCI-23L-LEC-1 Introduction to Laboratory and Scientific Methodol

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Table of Contents 1. Objectives 2. Introduction 3. Experiment: Biochemical Assay of Β-galactosidase 4. Epidemiology Results 5. The Objective of This Lab 6. Grading 7. Experimental Materials 8. Pre-experiment Preparation 9. Experimental Procedure 10. Cleanup 11. LS23L Safety Sheet for Biochemical Assay of β-galactosidase Activity Lab 12. Standard Operating Procedure: Lab C 1. Objectives Learn how to use the chromogenic substrate ONPG to assay β-galactosidase activity in E. coli. 2. Introduction Many types of enzymes (proteins that perform all sorts of functions), are found in cells. Some enzymes are needed all the time (e.g., hexokinase involved in glucose metabolism), whereas others are needed only under certain conditions. If all the enzymes that a cell will possibly need are produced all the time, the cell will waste a lot of energy. Therefore, there is a need to devise a mechanism to activate the transcription of an enzyme when this enzyme is needed, and to repress or shut down the transcription when this enzyme is not in demand. The enzyme β-galactosidase catalyzes the hydrolysis of lactose (a disaccharide found in milk) to galactose and glucose. Lactose is only one of the many energy sources that bacteria utilize. Bacteria growing in glucose (no lactose) have no need for the enzyme β-galactosidase. Is this enzyme still synthesized under these conditions? If so, how much? How can you tell? Lab Manual - Lab C - Beta-galactosidase Assay Return to top   Return to top  

Scientists have discovered that the transcription of β-galactosidase is regulated by the presence or absence of lactose in the environment (Figure C.1). Since the l gene in the lac operon is always active, its protein product, the repressor, is constantly being made. The repressor binds to the operator, prohibiting the lac operon genes from being transcribed by blocking the RNA polymerase. When lactose is present, it binds to the repressor, causing a conformational change of the repressor, and makes it fall off from the operator region. This allows the transcription of β-galactosidase, which later cleaves lactose. Enzyme activity is measured using a procedure called a biochemical assay. An assay is a method to indirectly assess how much of a given protein (enzyme) is produced per cell, per unit time. The ability to measure enzyme activity, like the ability to determine protein structure (introduced in the Epidemiology and Lab Techniques lab), is a technique critical to the investigation of the relationship between genes, proteins, and cellular function.

Figure C.1. Regulation of transcription of the lac operon in E. coli . When lactose is not present the lac-repressor protein which is always expressed and present (transcribed into mRNA & translated into protein) will bind to the lac Operator and block transcription of the Lac genes Z, Y and A. When lactose is present, it will bind to the lac-repressor and its conformational change will make it fall off the lac-operator. The transcription of the lac-genes will commence into mRNA, which in turn will be translated into protein. Figure C.2a. β-galactosidase reaction using lactose. β-galactosidase is an enzyme which hydrolyzes lactose into glucose and galactose. Glucose and galactose are colorless substances and are not easily detectable.

Figure C.2b. β-galactosidase reaction using ONPG as the substrate. When glucose is substituted with o-nitro-phenol, we get o-nitrophenyl-β-D-galactosidase (ONPG). This compound will also have a β-galactosidase linkage, which is chromogenic substrate for and will be hydrolyzed by β- galactosidase. The compound o-nitro-phenol has a yellow color, which can be easily measured in an assay. 3. Experiment: Biochemical Assay of Β-galactosidase To measure the amount of enzyme in a cell, one needs to determine the amount of that product produced in a given time by a given number of cells. Since it is not easy to assay β-galactosidase activity by measuring the amount of galactose or glucose produced or the amount of lactose consumed, a derivative of lactose, o-nitrophenyl-β-D-galactosidase (ONPG), is used as the substrate (Figure C.2). ONPG is a colorless compound. However, cleavage of ONPG by β-galactosidase produces a yellow compound, o-nitrophenol. This makes ONPG a useful chromogenic substrate for assaying β-galactosidase activity. The amount of o-nitrophenol produced is measured by determining the absorption (optical density, OD) of a sample at 420 nm in a spectrophotometer. The amount of o-nitrophenol produced per minute per bacterium reflects the amount of enzyme in a typical cell. The b-galactosidase assay was originally developed by a scientist named Jeffrey Miller, and therefore the units we use to measure b-galactosidase activity are called Miller units. For our purposes, units of b-galactosidase activity (Miller units) in this assay will be defined as: Units = OD / (time X volume X OD ) OD is the optical density (absorbance) of a sample at 420 nanometers and is proportional to the amount of o-nitrophenol produced. Time is the length of time that the reaction of β galactosidase and Return to top   420 600 420

ONPG is permitted to proceed; it is measured in minutes. Volume is the volume of the sample (removed from a culture) that is assayed for β-galactosidase activity; it is measured in milliliters. OD is the optical density of a sample at 600 nanometers; it provides an estimate of cell density in the sample (i.e., the number of cells/ml). (An OD equal to 1 represents approximately 2 X 10 cells/mL). Before you measure the optical density of any sample, you should measure a “blank” that provides the baseline optical density of the sample of interest. For example, to measure the optical density of bacteria cells, you should use the blank that contains Luria broth only (provided). To measure the optical density of o-nitrophenol, you have to prepare a blank containing all the ingredients except bacteria cells, glucose, and lactose. 4. Epidemiology Results In addition, during this lab you will have a chance to see and interpret your results from the Epidemiology and Lab Techniques lab exercise. Your TA will arrange your streaked plates, which were allowed to grow for 24 hours, on the side bench. During one of your incubations, you should work with your group to sketch out the plate growth and determine who was patient zero. 5. The Objective of This Lab In this experiment you will use the chromogenic substrate ONPG to assay β-galactosidase activity in E. coli cells grown in the presence of glucose and lactose, as well as in the absence of a sugar substrate (the control). The assay will allow you to determine: (1) if E. coli synthesizes the enzyme β- galactosidase in the absence of lactose, the substrate upon which β-galactosidase acts, and (2) if the induction of β-galactosidase synthesis in E. coli is time dependent. To accomplish this, you will assay β-galactosidase activity at two time points during the experiment: (1) 20 minutes after E. coli cells are introduced to sugar (glucose or lactose) and (2) 70 minutes after the introduction. A few notes about the protocol: Luria broth provides essential nutrients for bacterial growth. PopCulture Reagent is a buffered mixture of detergents that perforates the coli cell walls to release proteins into solution without denaturation. 600 600 8 Return to top   Return to top  

Z buffer promotes the reaction between β-galactosidase and ONPG by optimizing the pH of the sample. The hydrolysis reaction of β-galactosidase starts as soon as the substrate ONPG is added. Addition of Na CO (Sodium bicarbonate) stops the reaction of β-galactosidase and ONPG by changing the pH from 7.0 to 11. β-galactosidase is very pH sensitive. 6. Grading In addition to the individual quiz given at the beginning of the lab, each group will turn in a sheet showing their measurements of cell culture growth and β-galactosidase activity at the end of the lab. You will also work with your group to complete a worksheet on your plate data from the epidemiology lab. The worksheet will also include answers to specific questions regarding your results. 7. Experimental Materials Your TA Will Provide Ice Bucket Containing: Culture tubes LB labeled “A” Culture tubes LB and 4% glucose labeled “B” Culture tubes LB and 4% lactose labeled “C” Microcentrifuge tubes with 100 µl of LB labeled as “β-gal blank” Culture tubes MG 1655 coli cells Centrifuge tubes Z buffer mix Centrifuge tubes ONPG LB blank cuvette Centrifuge tubes 1 M Na CO Microcentrifuge tubes containing PopCulture Reagent NOTE: Reagents (except Na CO and PopCulture Reagent) must be kept on ice when not being used. 2 3 Return to top   Return to top   2 3 2 3

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