Biochemical Assay of β-Galactosidase: Lab C Manual Guide

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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.

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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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Other Materials and Their Locations Instructor’s Bench: Gloves Cuvettes Microcentrifuge tubes In the Fume Hood: Autoclave container for used tips and tubes Student Group Tray: 20 µl pipettor 200 µl pipettor 1000 µl pipettor Cuvette rack Microcentrifuge tube rack Test tube rack Sharpie Box of small tips Box of large tips Autoclave waste cup 8. Pre-experiment Preparation 1. Obtain the following test tubes from your TA: Test Tube A: 4.0 ml Luria Broth (LB). Test Tube B: 3.6 ml LB + 400 µl 4% glucose Test Tube C: 3.6 ml LB + 400 µl 4% lactose NOTE: The capital letters A , B , and C refer to these tubes throughout the remainder of the protocol. Return to top  

2. Obtain a total of six microcentrifuge tubes and thirteen cuvettes for each group from your TA. Label two microcentrifuge tubes as “a”; repeat for “b” and “c” for a total of six tubes. These tubes will be the subculture of tubes A, B, and C. Organize the thirteen cuvettes on the cuvette rack the same way as the microcentrifuge tubes. Label four cuvettes as “a”; repeat for “b” and “c” for a total of twelve cuvettes. The additional cuvette is for your β-gal blank. Make sure you write only in the upper area of a cuvette. 9. Experimental Procedure See the experiment flowchart ( Figure C.3 ), which has the same procedural labels. 1. Time Zero: Establish coli Cultures in Test Tubes A, B, and C a. Retrieve E. coli from your TA. b. Pipette 400 µl of E. coli bacteria into each A, B, and C tube. c. Gently mix the coli cells and broth by closing cap tightly and inverting each tube (A, B, and C) 1-2 times; tap the tubes to mix well. d. Label each tube (A, B, and C) with your group’s name; loosen caps for incubation. e. Place them in the 37°C incubator. Note the time. This is TIME ZERO. f. Incubate the tubes for 20 minutes. 2. Time 20 Minutes: Begin Analyses NOTE: Step 2a and 2b should be done concurrently by different members in the group. a. Subsample cultures A, B, and C to determine coli cell density: i. Retrieve one set of three cuvettes that have been labeled with a, b, and c. ii. Retrieve tubes A, B, and C from the incubator. Gently mix the E. coli cells and broth by closing cap tightly and inverting each tube (A, B, and C) 1-2 times. iii. Remove 1 ml from tube A and place it in the cuvette a. Repeat for tubes B and C. Proceed to Step 3: Determine E. coli Cell Density. b. Subsample cultures A, B, and C to assay cultures for β-galactosidase activity: i. Retrieve one set of three microcentrifuge tubes labeled a, b, and c. ii. Remove 100 µl of cell culture from tube A and add it to microcentrifuge tube a. Repeat for tubes B and C. iii. Return culture tubes A, B, and C to the incubator for the 70-min assay. iv. Pick up PopCulture Reagent and a “β-gal blank” (containing 100 µl of LB) from your TA. Add 10 µl of PopCulture Reagent to tube a. Repeat for tubes b, c, and the “β-gal blank”. Vortex all tubes for 30 seconds, then allow to sit at room temperature for 10 minutes. v. Proceed to Step 4: β-galactosidase Assay. Return to top  

3. Determine E. coli Cell Density a. Follow directions posted on the spectrophotometer. b. Be sure that the spectrophotometer is set to read at 600 nm. c. Use the LB blank provided (NOT the “β-gal blank” that is incubating with PopCulture). d. Measure the absorbance (optical density, OD) of the 1 ml subsamples labeled a, b, and c that you placed in the cuvettes in Step 2a. e. Record your data (the values in the column headed 600 λ). f. Discard subsample cuvettes into the autoclave waste cup; leave the LB blank for other groups. 4. β-Galactosidase Assay a. When the 10-minute PopCulture incubation is done, vortex all 4 microcentrifuge tubes for 30 seconds. b. Pick up Z-buffer from your TA. Add 600 µl Z-buffer to tube a. Repeat for tubes b, c, and the “β- gal blank.” c. Add 140 µl of ONPG (4 mg/ml) to each of the three samples AND the “β-gal blank.” Mix by inverting each tube several times. d. Allow tubes to sit at room temperature for 15 minutes. During this time β-galactosidase, if present, will cleave ONPG to produce the yellow product o-nitrophenol. e. Add 350 µl of 1 M Na2CO3 to each of the three samples AND the “β-gal blank.” Mix by inverting each tube several times. Na2CO3 stops the cleavage reaction (yellow color is stable after the addition of Na2CO3). f. Transfer 1 ml of sample a into a cuvette labeled “a.” Repeat for samples b, c, AND the “β-gal blank.” g. Measure the absorbance (optical density, OD) of a, b, and c at 420 nm using the “β-gal blank” as your blank. Record your data (the values in the column headed 420 λ). h. Save the “β-gal blank” for the Time 70 analysis. i. Discard sample cuvettes a, b, and c into the autoclave waste cup. 5. Time 70 Repeat the steps above, starting at Step 2 (Time 20), to measure cell growth and β-galactosidase activity 70 minutes after time zero

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Figure C.3. Experiment flowchart. Illustration of treatments to tubes B and C from step 2 are omitted as they are handled exactly the same as tube A. Click here to open a larger version of Figure C.3 . (https://bruinlearn.ucla.edu/courses/178461/files/15508736?wrap=1) Eppendorf Biospectrometer Operating Instructions At the beginning of each section: The spectrophotometer will be on already, selected for “Beta-gal,” and 420 and 600 nm.

Before placing a cuvette in the spectrophotometer: Use a Kimwipe to wipe down the sides. If bubbles are visible, tap gently or cover and invert. The side of the cuvette with the arrow should be facing the arrow on the spectrophotometer. For each group: Insert the appropriate BLANK cuvette for your group and the reading you are taking, close the cover of the spectrophotometer. Press “Blank” at the bottom right of the spectrophotometer. Remove the BLANK cuvette once the machine has finished reading. To read samples: Insert SAMPLE cuvette, close the cover of the spectrophotometer. Press “Sample” at the bottom right of the spectrophotometer. Record absorbance number displayed for your wavelength of interest and remove SAMPLE cuvette. Repeat steps 4–6 for the remaining samples. At the end of each section: Close the cover on the spectrophotometer. 10. Cleanup Autoclave waste cup—empty into the autoclave waste bag in the hood and replace cup at station. Used culture tubes (A, B, C)—place tubes with contents in the autoclave waste bag in the hood. Return all items to their original places and clean up your station. 11. LS23L Safety Sheet for Biochemical Assay of β-galactosidase Activity Lab You must review this sheet and the attached SOPs carefully before coming to lab. You are responsible for following all safety guidelines while in lab and you will be tested on this material in the pre-lab quiz. Return to top   Return to top  

Standard Operating Procedures (SOPS) Referred to in This Lab E. coli strain K-12 MG1655 Potential Hazards E. coli strain K-12 MG 1655 is a debilitated strain of bacteria that is not normally considered pathogenic in healthy adults but young, elderly, immunocompromised individuals, or individuals taking large quantities of antacids or stomach acid reducing medications are at higher risk of infection. If infected, inflammation, diarrhea, or conjunctivitis may occur. Engineering and Work Practice Controls E. coli cultures should only be used at the lab benches and in the hood, as directed in the lab manual. Avoid any contact with mucous membranes as they are the most common route of entry. Wash hands before leaving the lab. Personal Protective Equipment (PPE) In addition to standard lab attire, you must wear: Safety glasses or goggles for the entire lab. Gloves when working with any chemicals or cell cultures. Change gloves as necessary. Lab coat for the entire time you are in the lab room. Waste Disposal Microcentrifuge tubes, pipette tips, cuvettes, and culture tubes contaminated with E. coli must be placed in the autoclave bag provided in the fume hood.

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12. Standard Operating Procedure: Lab C Chemical name: E. coli strain K-12 MG1655 CAS #: N/A PI: Dr. Gaston Pfluegl Updated: 6/18/2019 Building: Young Hall South Room(s): YHS 2344, 2336, 2370, 2379 1. Circumstances of Use/Storage LS23L uses the K-12 MG 1655 strain of E. coli in the Biochemical Assay of β-Galactosidase Activity lab. E. coli strains must be handled safely in accordance with UCLA policy and standard microbiological methods. E. coli K-12 is considered a Risk Group 1 (RG1) agent and is not associated with disease in healthy adult humans. 2. Potential Hazards E. coli K-12 is not considered a human or animal pathogen nor is it toxicogenic. The strain E. coli K-12 is a debilitated strain which does not normally colonize the human intestine. It has also been shown to survive poorly in the environment, has a history of safe commercial use, and is not known to have adverse effects on microorganisms or plants. Although organisms classified as Risk Group 1 are not known to consistently cause disease in healthy adults, the following individuals may be at increased risk of infection: young, elderly, immunocompromised individuals, or individuals taking large quantities of antacids or stomach acid-reducing medications. Inflammation may occur if autoinoculation results from injection or contamination of broken skin. Ingestion of E. coli may cause diarrhea with or without abdominal cramps. Skin infections may be inflamed. Conjunctivitis may occur after inoculation of the eye. 3. Engineering Controls N/A Return to top  

4. Work Practice Controls E. coli cultures should only be used at the lab benches and in the hood, as directed in the lab manual. Avoid any contact with mucous membranes as they are the most common route of entry. Wash hands before leaving the lab. 5. Personal Protective Equipment (PPE) All personnel are required to wear the following personal protective equipment whenever handling this material: Safety glasses or goggles, gloves, lab coat or apron, and closed shoes (absolutely no sandals, open-toed or open-heeled shoes). 6. Transportation and Storage E. coli cultures are grown in the prep room (YHS 2344) and small amounts are transported to the lab rooms (YHS 2336, 2370, and 2379) in culture tubes on ice. E. coli plates are kept in the refrigerator in YHS 2344. 7. Waste Disposal LS Core lab staff is responsible for the safe collection, preparation, and proper disposal of waste. Microcentrifuge tubes, pipette tips, and culture tubes contaminated with E. coli must be placed in the autoclave bin provided in the fume hoods of the lab rooms (YHS 2336, 2370, 2379). Prep staff will collect all waste from the bins and decontaminate in the autoclave. 8. Exposures/Unintended Contact UCLA Campus Police—911 from campus phone or 310.825.1491 from cell Environmental Health and Safety—x59797 (310.825.9797) Emergency Room (Ronald Reagan Medical Center)—x52111 (310.825.2111)

Complete the incident report found on the LS Core Labs prep server and notify EH&S of any serious accidents within 8 hours (310.825.9797). Skin Contact: Wash area thoroughly with soap and water for up to 15 minutes. Eye Contact: Immediately flush eyes with plenty of water for at least 15 minutes, lifting upper and lower eyelids occasionally. Inhalation: Remove to fresh air. If breathing is difficult, give oxygen. Get medical attention. Ingestion: Seek medical attention. 9. Spill Procedure All large spills must be reported to EH&S (310.825.9797) as soon as possible. Use personal protective equipment as described above. LARGE SPILLS: Mix spill with bleach to get 10% final concentration, allow decontamination for 30 min. Adsorb spill with paper toweling and remove to autoclavable tray or bucket. Wipe spill area clean using 10% bleach. SMALL SPILLS: Decontaminate surfaces with 70% ethanol or 10% bleach. 10. Authorized Personnel LS Core Lab Staff LS Core TAs and LAs (only after TA/LA training meeting) LS Core Students (only under supervision of trained TAs) 11. Required Training LS Core Lab Staff training should include the following: Review of the current MSDS Review of the departmental safety procedures Review of the UCLA Chemical Hygiene Plan Completion of the UCLA Laboratory Safety class administered by EH&S Completion of annual online refresher Laboratory Safety class administered by EH&S

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Completion of annual PPE training Return to top 

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