Enzyme Induction in a Prokaryote Protein expression in Escherichia coli is an important and widely used method for studying the functions of enzymes involved in cellular pathways that affect the behavior and homeostasis of cells.
Lab 4 Enzyme Induction in a Prokaryote Protein expression in Escherichia coli is an important and widely used method for studying the functions of enzymes involved in cellular pathways that affect the behavior and homeostasis of cells. Examples of some of these cellular pathways include cell signaling, metabolism, cell cycle regulation, and secretion, among many others. The information gleaned from protein expression studies have the potential to provide profound information on cellular workings. For example, this type of research can elucidate the mechanisms of disease, ultimately leading to the discovery of better therapeutic drugs. In another example, a clear understanding of a particular cellular pathway may affect change in public policies or attitudes regarding the environment or conservation of endangered species. For our purposes today, we will focus on a small example of cell signaling in the bacterium, Escherichia coli, and a single, specific protein pathway. First, it is important to understand cells contain thousands of different genes, each coding for a different product. For example, E. coli cells contain over 4300 genes. If all these genes were expressed, or turned on, at the same time, the cell would be overwhelmed, would very quickly run out of energy, and would die. Therefore, it is not surprising to learn that cells are extremely selective about which genes are active or expressed in a given moment, and in what amounts. This is the model system we will use to look more closely at enzyme induction. The specific genetic model system we will investigate is the metabolism of lactose by E.coli. Lactose is a disaccharide made of two monosaccharides – galactose and glucose. For a cell to use lactose as an energy source, it must be able to break the sugar down into its monosaccharide subunits. The enzyme responsible for this reaction is β-galactosidase, and the reaction it catalyzes is shown in Figure 1: Figure 1. Catabolism of Lactose using the beta-Galactosidase Enzyme. Investigation of this system by Jacques Monod and François Jacob in the 1950s was among the earliest research into cell regulation of gene expression. Jacob and Monod observed that when E. coli cells are switched to a medium containing lactose, they begin to synthesize three proteins involved in the assimilation and metabolism of lactose. One of these is β-galactosidase. They also demonstrated that a regulator gene controls the transcription of the genes encoding the lactose metabolism proteins. According to their model, the regulator gene encodes a repressor protein that binds the DNA upstream of the lactose metabolism genes. When the repressor is bound to the DNA, it prevents RNA polymerase from transcribing those genes. To explain how the E. coli can express (or turn on) these genes in the presence of lactose, they hypothesized that lactose interacts with the repressor protein, causing it to release the DNA, and that this release allows the lactose metabolism genes to be transcribed. Their experiments showed that lactose binds to a specific site on the repressor protein, changing its shape in such a way that its DNA binding activity is inactivated. Molecules that trigger the expression of genes are called inducers. Inducers are natural to cellular pathways. If lactose is present in a natural bacterial environment it acts as an inducer to start the expression of lactose metabolism genes in E. coli. This method of gene regulation means that the proteins required for lactose assimilation and metabolism are only produced when cells are exposed to lactose. If lactose is not in the environment, cells do not waste energy transcribing and translating genes they do not need. When all available lactose is used up, the lactose metabolism genes are turned off again by the repressor protein. An inducer is like an activator or initiator that starts something happening – in this case, expression of an enzyme. The enzyme can then act on the substrate to produce a product. The chemical reaction is shown below. In this assay, the inducer is lactose, the enzyme is ß-galactosidase, the substrate is ONPG (o-nitrophenol galactose), and the products are o-nitrophenol (which makes the yellow color) and galactose. If lactose is present in the growth medium when used as the inducer, ß-galactosidase enzymes will be engaged in breaking down lactose substrates to produce glucose and galactose.
IPTG is a synthetic chemical that mimics the action of lactose. It is often used in molecular biology research to induce the expression of a protein that is the focus of a study (we will revisit this concept during our cloning project). Figure 2. Catabolism of ONPG via beta-Galactosidase OBJECTIVE In this experiment, students learn about the induction of protein expression in an E. coli system. Students collect data on the protein’s function by assaying for the enzyme activity of two E. coli strains after they have been induced with lactose or IPTG. A control reaction is run in tandem, using an equal volume of water instead of an inducer. The induction experiment is done on two different E. coli strains for comparison of enzyme activity after induction. One strain has the genotype lac+ and carries the gene for the b-galactosidase enzyme. The other strain has the genotype lac-, and has this gene deleted. Cell extracts from 6 culture conditions will be assayed by different groups in class: Lac+ induced with lactose, Lac+ induced with IPTG, Lac- induced with lactose, Lac- induced with IPTG, and Lac+ and Lac- controls (see Table 1). Each group of students will collect absorbance data for their assigned set. Student data is reported in class and pooled so that each student can include data for all six culture conditions in their results and analysis for comparison. Students calculate and graph the rates of induction over time for all six sets of strain inductions. OVERVIEW OF THE METHOD The Lac+ and Lac- strains were grown and induced with either lactose or IPTG. Control cultures were grown at the same time. Cell extracts were prepared from samples taken at a series of timepoints after induction. For this lab, the cell extracts will be provided for each student to assay. Put another way: the induction and collection of extracts at specific time points has already been done. Your part of the lab is to determine enzyme activity from these pre-made extracts. Enzyme activity is assayed by the addition of ONPG substrate and incubation in a 28°C heat block for development of a yellow color produced in the reaction. The reaction is stopped by adding sodium carbonate, Na2CO3, and the resulting absorbance is measured in the spectrophotometer at OD415, The relative enzyme activity and concentration of o-nitrophenol for each sample will be calculated using the standard curve for p-nitrophenol that you made in a previous lab. MATERIALS AND EQUIPMENT ¥ 5 E. coli cell extracts; timepoints are minutes after induction: 0, 30, 60, 90,120. ¥ Z buffer ¥ o-nitrophenol galactose (ONPG) stock solution, 4 mg/mL ¥ sodium carbonate (Na2CO3) stock solution, 1 M ¥ 1.7 mL microtubes ¥ 2 cuvettes ¥ Spectrophotometer ¥ 28°C heat block
PROCEDURE Students work in pairs or groups of three. Each group will be assigned one set of cell extracts containing timepoints after induction for a strain or a control by the instructor. Timepoint sets are one of six strain/conditions: Table 1: Summary of Timepoints, Inducers and Lac strains Table 2: Preparation of Extracts Reagent: Volume: Cell extract 25 µL Z Buffer ONPG, 0.25 mg/mL final concentration Total: 400 µL *Have your instructor verify your totals before you proceed!
PROCEDURE, continued: 1. Calculate your reaction volumes and have these volumes verified by your instructor. 2. Add your selected cell extracts to microcentrifuge tubes. 3. Next, add Z buffer to each cell extract timepoint and equilibrate the temperature by incubating in the heat block at 28°C for 5 minutes. 4. Before you start the reaction, make sure that the duties are divided up amongst group members. You will need to record the exact time (to the second) that the reagents are added to start and to stop the reaction. Thus, one person should act as the scribe, one person should be the time keeper and a third person can add the reagent(s). 5. Initiate the reactions by adding ONPG and record the exact clock time of addition (this is the time of initiation or t0) to the nearest second. 6. Return to incubation at 28°C. Your tubes should not sit in the incubator for longer than 10 minutes. 7. Stop the reactions by adding 600 uL of sodium carbonate into the tube, recording the exact clock time (this is the stop time or tstop) to the nearest second. 8. Cap the tube securely. Vortex briefly to mix well, then place the tube in a rack to hold at room temperature. Measure the absorbance at 415 nm for each reaction. 9. Calculate the amount of time of each reaction in minutes between the initiation (t0) and the stop (tstop). This is the number of minutes between the addition of ONPG to the addition of sodium carbonate. The relative enzyme activity is calculated using the equation: Relative Enzyme Activity = A415___ tstop-t0 (min) Use the equation from your p-nitrophenol standard curve in Lab 3 to calculate the amount in micromoles (umol) of o-nitrophenol produced in each reaction by entering the absorbances in this experiment. Enter all your data into Table 3 and report your relative enzyme activity (A415/ tstop-t0) numbers and the reaction product o-nitrophenol (o-NP) concentrations for each time point to your instructor. Your instructor will gather the class data and post the results on the whiteboard and/or online. Everyone should use the class data in their lab report to analyze and compare the activity of β-galactosidase in induced vs. uninduced cultures of a Lac+ and a Lac- strain. Table 3. Data for Enzyme Induction Experiment Sample time point Assay clock times tstop-t0 (min) A415 measured A415 tstop-t0 [o-NP] (umol) ONPG added (t0) Na2CO3 added (tstop) Example 12:00:00 12:06:30 6.5 min 0.455 0.07 3.7 0 30 60 90 120
WRAP UP, CLEAN UP AND LAB NOTEBOOK: ¥ Your post lab activity will use the class data you collected in lab today and you will need to use your standard curve from Lab 2, so make sure you have the information needed. Waste Item Proper Disposal Microcentrifuge Tubes Empty liquid into waste beaker. Cuvettes Empty liquid into waste beaker. Rinse well with DI H2O and place in “Cuvette Drying Rack” to dry. Waste Beakers Please empty in the labeled liquid waste container. Wash well, replace to dry. ¥ Please return all reagents to their original location! ¥ Please disinfect your workspace and put all instruments back in the correct location. ¥ Don’t forget to wash your hands before you leave. In Your Lab Notebook: Report the results of all your experiments in scientific format, stating: Hypothesis -what do you predict will happen in these experiments, in an if/then statement(s) Purpose – what are you learning/accomplishing in these sets of experiments/ Materials and Methods – what did you use and how did you use it? Results – this is where you include your completed data tables, calculations, and graphs (these may be completed on the computer and pasted into your notebook if you prefer but must be completed by you) Discussion and Conclusion – This is where you can interpret your results and you can address any problems you had along the way. Note that in this lab, you likely did not complete all the exercises, rather the data was gathered by different groups and shared. You need to have all the data in your lab notebook and interpretations of the results as directed by your instructor.
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