Lac Operon: Turning On Your Genes Activity Kit 47384

KIT CONTENTS
Item 1 2 3 4 5 6 7 8 9 10 Quantity/Volume 1 box 3 feet 3 feet 1 roll 15 1 package 1 package 1 package 1 1 Description Colored metal paper clips Tubing, 1/4 Tubing, 5/16 Red electrical tape Dowels with labels Yellow paper White paper Orange paper Instructional video Set of instructions (this booklet): Teachers Guide: Pages 1 - 7 Student Guide (reproducible): Pages S1 S7

Additional materials needed: transparent tape scissors permanent markers rulers trays or shoe boxes, for storage, distribution of lab materials (optional)

INTRODUCTION
At the time of conception, a single-celled fertilized human egg contains approximately 100,000 genes. However, the genetic information contained in these 100,000 genes is not all expressed at the same time. Genes are turned on (transcribed and translated) and turned off throughout the life of the organism at appropriate times. For instance, genes that direct the development of fingers and toes in humans are on for a brief time during early development. After a fetus develops fingers and toes, the gene products needed for digit production are no longer needed. These genes are turned off and remain off for the rest of the persons life. An operon is a section of DNA responsible for turning on and turning off the genes at specific times. An operon contains structural genes (genes that code for polypeptides), a promoter (initiation site where transcription begins), and an operator (the binding site for the repressor protein). Repressor proteins prevent transcription of mRNA by RNA polymerase by binding to the operator. The lac operon is the operon that allows the body to metabolize and utilize lactose (absorbed from the intestinal environment) as a source of energy. In the presence of lactose, the lac operon is turned on and the Lac Z, Lac Y, and Lac A enzymes coded for by genes on the lac operon can be synthesized and the lactose can be metabolized. In the absence of lactose, the transcription and translation of these genes is repressed or turned off. In this lab, students are given background information on gene expression. Students will then use a simple model to illustrate how and when the lac operon is turned on and off. A worksheet is also included, to reinforce and test students understanding of concepts discussed and demonstrated in the lab. This kit includes enough materials for 15 student lab stations. Students may work individually or in small groups. p.1
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TIME ALLOTMENT
The lab activity (including review of background information and performing the student activities) requires approximately 40 minutes to complete. The worksheet questions can be assigned as homework or completed during another class session.

LAB OBJECTIVES
Using this kit, your students will: Manipulate a model of gene regulation and expression. Explain how it is possible for somatic cells to develop different characteristics and functions. Describe gene regulation in prokaryotic cells. Describe transcription factors and enhancers, promoter, regulator, and operator regions in the transcription of a gene. Explain how the presence or absence of lactose affects the lac operon.

ASSEMBLING THE KIT

The first time you use this kit, you will need to assemble the following pieces. These pieces should be collected at the end of the lab, so that they can be re-used. NOTE: Please reveiw the video to identify the following pieces. Repressors 1. Cut 15 - 2.0 lengths of 1/4 tubing 2. Make a full-length slit along one side of each 2.0 tube, cutting all of the way through the plastic on that side. With the point of the scissors, pierce a small hole in the wall of the tubing across from the slit, 1 from the end. Using a permanent marker, label each of these tubes with the word repressor. RNA polymerase 1. Cut 15 - 1.5 inch lengths of 5/16 tubing. On each piece of tubing, wrap a 3/4 wide piece of red electrical tape around one end, so that it covers half of the tube. Do not cover either open end of the tube with the tape. Using a permanent marker, label each of these tubes with the words RNA polymerase. Lactose / Inducers 1. Unfold the last bend in each paper clip. The straight portion of the paper clip will stick through the repressor tube as shown.
red tape

2" clear tubing with slit down its length.

Unfolded paper clip is pushed through the tube so it can't be applied over the operator.

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Permease** (see ** note below) From the orange construction paper, cut 15 triangles, each no larger than 1.5 inches at its widest point. Label each triangle with the word lac permease. -galactosidase** (see ** note below) From the yellow construction paper, cut 15 - 1 x 1 yellow squares. Label each square with the word -galactosidase. Acetylase** (see ** note below) From the white construction paper, cut 15 - 1 diameter circles. Label each circle with the word acetylase. ** Since these are so easily and quickly produced and plenty of materials are supplied for them, you may prefer to have each group of students construct their own permease, galatosidase, and acetylase shapes at the beginning of class. Save the extra paper in case pieces are lost.

PRIOR TO CLASS
1. Review the videotape included with this kit prior to performing this activity. 2. Read through the Student Instructions (pages S1 - S7), to familiarize yourself with this activity. 3. If desired, assemble all lab materials in individual trays or boxes for easy pick up and return. Materials needed per Lab Station 1 labeled dowel (lac operon) 1 RNA POLYMERASE tube 1 split REPRESSOR tube 1 -galactosidase yellow square

1 permease orange triangle. 1 acetylase white square. 1 lactose/inducer paper clip (first bend straightened) Transparent tape (not included in kit)

SUGGESTED LESSON PLAN

1. With students, review transcription, translation, gene expression, relevant vocabulary, and the role of the lac operon in lactose metabolism. Background information is provided in the Student Guide. 2. Give students a brief overview of the lab activity. 3. Distribute lab materials. 4. Show students which items in this lab investigation represent the operon, RNA polymerase and so forth. (Refer to the list below.)

lac operon labeled dowel promoter green section of dowel regulator blue section of dowel operator red section of dowel RNA polymerase clear tubing with red tape repressor protein clear tubing split lengthwise lactose / inducer unbent paper clip
5. Have students perform the lab activity. For better understanding, you may wish to have students repeat parts B and C of the lab several times.

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6. After completion of the lab, collect all lab materials. 7. Have students complete worksheet questions and research, either as an in-class activity or homework.

LABORATORY EXTENSION
Use the Science Kit Regulation of Gene Expression kit (#46962) to complement the Lac Operon Activity.

RESEARCH EXTENSIONS
1. Visit the following website, and work through the tutorial and problem sets. http://www.biology.arizona.edu/molecular_bio/problem_sets/ mol_genetics_of_prokaryotes/prokaryotes/htm 2. Explain why lac operon is off when both glucose and lactose are present together in the environment. 3. Explain how -galactosidase in E. coli differs from lactase in Homo sapiens. 4. What evidence is there that the production of human lactase is inducible/repressible? 5. Describe the model for eukaryotic transcription. Include a description of how a polymerase binds and how proteins binding to the promoter and enhancer elements act. 6. Proteins involved in eukaryotic gene regulation are often modified. Describe how and why.

REFERENCES
Johnson, George B., Raven, Peter H., 1998. Biology: Principals and Explorations. Holt, Rinehart, and Winston, Harcourt Brace, and Company. Orlando, Fl. Pp. 190-191. Hallick, Richard B., Regulation of Inducible Genes. [Online]. Available p. 2. http:// www.blc.arizona.edu/courses/181gh/rick/expression1/inducible/html, October 18, 1999. Knight, Stefan., BK2 Biochemistry 1997 Exercises., [Online]. http://alpha2.bmc.uu.se/ ~stefan/Courses/Bke2_1997/Exercises/Exercise11.html., 1997 The Biology Project. Molecular genetics of Prokaryotes. [Online]. Available http:// www.biology.arizona.edu/molecular_bio/problem_sets/mol_genetics_of_prokaryotes/ Prokaryotes.html Science Projets.com., Controlling a Gene 2; Kinetics of the Induction of the Lac Operon. [Online]. Available http://www.science-projects.com/beta-galase.htm Ingram, Vernon, MIT Biology Hypertext Book, The Lac Operon. [Online]. Available http:// esg-www.mit.edu:8001/bio/pge/lac/html Kimball, John W., Positive Control of Transcription: CAP., [Online]. Available http:// www.ultranet.com/~jkimball/BiologyPages/L/LacOperon.html p. 4
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Schleicher, Phil, Thomas, Ron, transformation of E.Coli, Production and Assay of -galactosidase by an Aerobic Fermentation. [Online]. Available http://www.uwrf.edu/biotech/ workshop/activity/act14/act14.htm, Summer 1996. Essandoh, Nelly. Transcriptional Control in Bacteria: Lac Operon, Trp Operon, and catabolite repression. [Online]. Available http://www.dartmouth.edu/~cbbc/courses/bio23/bio231998/lecture-notes/05-TranscContBacteria.html, July 9, 1998 McGill University, The Lac Operon in E. Coli-notes.[Online]. Available http://www.mcgill.ca/ nrs/regpro-n.htm, 1999. Campbell, Neil A. 1996. Biology. Benjamin/Cummings Publishing Company, Inc.. Menlo Park, California. Pp.344-349.

ANSWER KEY
1. What is an operon and where is it located? An operon is a section of DNA that contains structural genes and an operator which is responsible for turning these genes on and off. Operons are located within the chromosomes of an organism.

2.

Draw the lac operon and its genes. Label the following structures: operator, regulator, structural genes, and promoter. Briefly describe the function of each structure.

(blue)

(green)

(red)

(yellow)

(orange)

(white)

regulator

promotor operator

Lac Z

Lac Y

Lac A

mRNA + ribosomes

galactosidase

permease

acteylase

Operator - the on/off switch for transcription of DNA into mRNA; binding site for the repressor. Regulator - this gene codes for the repressor protein. Promotor - binding site for RNA polymerase. Structural genes - Lac Z. Lac Y, and Lac A - These genes code for enzymes necessary for the metabolism of lactose.

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3. Draw an operon as it appears when it is turned on. Label all structures not labeled in question 2.
RNA polymerase
(blue) (green) (red) (yellow) (orange) (white)

regulator

promotor operator

Lac Z

Lac Y

Lac A

blocked repressor change in shape mRNA + ribosomes

lactose

galactosidase

permease

acetylase

4. Draw an operon as it appears when it is turned off. Label all structures not labeled in questions 2 and 3.
RNA polymerase binds but cannot move to transcribe RNA polymerase
(blue) (green) (red) (yellow) (orange) (white)

regulator

promotor operator

Lac Z

Lac Y

Lac A

repressor

No mRNA and no protein (enzymes)

5.

Explain how the lac operon is involved in the regulation of protein synthesis. The lac operon turns on the structural genes that direct the production of enzymes needed to metabolize lactose when it is present in a cells environment and is needed for energy. The lac operon also turns off those structural genes when lactose is absent from the cells environment or is not needed for energy.

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6.

What is the biological significance of operons? Operons play a significant role in the maintainance of homeostasis within a cell or organism. Without operons to turn genes on and off, genes would be on all the time. Organisms would become overloaded with products that are unnecessary or damaging in large amounts.

7.

Define transcription and translation and explain their relationship to each other. Transcription is the process in which a strand of DNA is copied into a complementary strand of mRNA. Translation is the process of reading the mRNA strand and using the information to assemble a chain of amino acids in the correct sequence. This amino acid string will become a protein. Transcription makes an mRNA strand (a copy of the DNA that is readable by tRNA) and the mRNA codons are translated and assembled into the amino acids of a protein on the ribosome.

8. Lac I codes for the repressor protein. Describe the effect on a cells regulation of galactosidase if lac I did not exist in that cell or became mutated and could not make a functional repressor? In the event that a functional repressor did not exist, mRNA transcription could not be prevented and the gene could not be turned off. -galacctosidase would be made all of the time whether lactose was present or not. The result would be an unnecessary expenditure of the cells energy and resources.

Carol Bullock Science Teacher Danbury High School Danbury, TX

2001, SKBL Printed in U.S.A. SK04546-00

Tonawanda, NY/San Luis Obispo, CA 1-800-828-7777 St. Catharines, Ontario, Canada 1-800-387-9393 www.sciencekit.com

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Lac Operon: How To Turn Genes On! Student Guide

VOCABULARY
Operon section of DNA responsible for turning on and turning off specific genes at specific times. An operon contains structural genes (genes that code for polypeptides), a promoter (initiation site where transcription begins), and an operator (the binding site for the repressor protein). Lac operon (entire labeled dowel) operon that allows the body to metabolize and utilize lactose (absorbed from the intestinal environment) as a source of energy. Promoter (green portion of dowel) a sequence near the start of a gene where RNA polymerase attaches. Regulator (blue portion of dowel): a gene that codes for the repressor protein. Operator (red portion of dowel): the on/off switch for transcription of the DNA into mRNA. This is the gene region to which the repressor protein binds when lactose is not present or is not needed. RNA polymerase (clear tubing with red tape): enzyme that reads DNA and transcribes DNA into mRNA Repressor protein (clear tubing split lengthwise): a protein that binds to the operator and prevents RNA polymerase from binding to the promoter Lactose/inducer (paperclip): binds to the repressor to produce a change in the repressor, thus preventing the repressor from binding to the operator

INTRODUCTION
At the time of conception, a single-celled fertilized human egg contains approximately 100,000 genes. However, the genetic information contained in these 100,000 genes is not all expressed at the same time. Genes are turned on (transcribed and translated) and turned off throughout the life of the organism at appropriate times. For instance, genes that direct the development of fingers and toes in humans are on for a brief time during early development. After a fetus develops fingers and toes, the gene products needed for digit production are no longer needed. These genes are turned off and remain off for the rest of the persons life. In order for cells to be as efficient as possible, each cell needs to be able to: Control the amount of gene products produced. Some products, such as ribosomal proteins, are needed in large amounts; most enzymes, required for reactions, are only needed in small amounts. If genes were not regulated, cell energy would be wasted. Respond to the environment by turning on or turning off specific genes or groups of genes. Turn genes on or off in the correct temporal pattern for proper growth and development, both before and after birth.

The most direct way to control the expression of a gene is to regulate the rate at which RNA polymerase transcribes the DNA in the gene into molecules of mRNA. The mechanism for control of gene expression was described by Francois Jacob and Jacques Monod, who discovered the operon model in 1961. An operon contains the structural genes, the operator, the regulator, and the promoter. The entire stretch of DNA required for enzyme production is referred to as an operon.

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(blue)

(green)

(red)

(yellow)

(orange)

(white)

regulator

promotor operator

Lac Z

Lac Y

Lac A

mRNA + ribosomes

galactosidase

permease

acteylase

Figure 1

When E. coli is in an environment that contains lactose, a group of genes found on the lac operon allow the bacterium to use the lactose as a source of energy. RNA polymerase can bind to the promoter, move across the operator, and transcribe the structural genes of lac Z, lac Y, and lac A. As a result; permease (which is coded for on the lac Y gene) allows lactose to be transported into the cytoplasm. Galactosidase (from the lac Z gene) metabolizes the lactose into galactose and glucose. Glucose is then used as an energy source. The role of acetylase (the product of lac A) is unclear.

C 12 H 22 O 11

H2O

C 6 H 12 O

C 6 H 12 O 6

Lactase

Lactose

Water

Galactose

Glucose

Figure 2

When lactose is present, it acts as an inducer, or an on switch. Lactose enters the cells and binds to the repressor. This binding causes a conformational change that allows the repressor to fall off the DNA. Now RNA polymerase is free to move along to transcribe the DNA of the three structural genes into mRNA. The mRNA is translated and the enzymes polymerase, b-galactosidase, and acetylase are synthesized. Lactose can then be metabolized. See figure 3. p. S2
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HO

HO

HO

HO

H H

O HO H HO H HO O HO H H H HO H

HO H
2

O H

HO

H HO H

O HO H H H O H HO O H HO H HO H H HO H

HO H

RNA polymerase
(blue) (green) (red) (yellow) (orange) (white)

regulator

promotor operator

Lac Z

Lac Y

Lac A

blocked repressor change in shape mRNA + ribosomes

lactose

galactosidase

permease

acetylase

Figure 3 When lactose is removed, the repressor returns to its original shape and binds to the DNA, so that RNA polymerase can no longer get past the promoter. No RNA and no proteins are made. See figure 4.

RNA polymerase binds but cannot move to transcribe RNA polymerase

(blue) (green) (red) (yellow) (orange) (white)

regulator

promotor operator

Lac Z

Lac Y

Lac A

repressor

No mRNA and no protein (enzymes)

Figure 4

LAB OBJECTIVES
As part of this lab you will: Manipulate a model of gene regulation and expression Explain how it is possible for somatic cells to develop different characteristics and functions. Describe gene regulation in prokaryotic cells. Describe transcription factors and enhancers, promoter, regulator, and operator regions in the transcription of a gene. Explain how the presence or absence of lactose affects the lac operon. p. S3
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PROCEDURE
Before starting this lab, make sure you have all of the following materials. 1 labeled dowel (lac operon) 1 RNA POLYMERASE tube 1 split REPRESSOR tube 1 -galactosidase yellow square* 1 permease orange triangle* 1 acetylase white square* 1 lactose / inducer paper clip (with first bend straightened) Transparent tape

*NOTE: If these items are not included, prepare the permease, -galactosidase and acetylase shapes, as directed by your teacher. PART A: Turning genes on! If lactose is present in the system and is needed for energy, somehow the genes on the lac operon must be turned on in order to produce the enzymes needed to metabolize the lactose. How is this done? Simulate the process as follows: Step 1: Lay the operon on the table with the REGULATOR (blue end) to your left (see Figure 5) Place each paper shape under its corresponding (same color) lac gene on the operon. For example, place the yellow square under the yellow lac Z gene.
(blue) (green) (red) (yellow) (orange) (white)

regulator promotor operator

Lac Z

Lac Y

Lac A

Figure 5

Step 2: Slide the RNA polymerase over the left end of the operon. Slide the RNA polymerase along the DNA operon model from left to right. See figure 6. This action simulates the transcription of the DNA. The lac Z, lac Y, and lac A (green) (red) (yellow) (orange) (white) (blue) genes will be tranLac Y Lac A regulator promotor operator Lac Z scribed and translated and their RNA polymerase respective enzymes (-galactosidase, permease, and acetylase) will be made. Figure 6 Step 3: After RNA polymerase has transcribed the entire operon, slide (green) (red) (yellow) (orange) (white) (blue) the colored pieces Lac Z Lac Y Lac A regulator promotor operator of paper out from RNA polymerase under the operon model and toward you. See figure 7. yellow orange red At this point, lactose can be tape together metabolized into using clear tape galactose and glucose. Figure 7 p. S4
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PART B: Turning off genes Step 4: Place the colored pieces of paper back under the operon. When lactose is not present, the lac Z, lac Y, and lac A genes do not need to be transcribed because -galactosidase, permease, and acetylase are not needed to break down lactose. In other words, NO LACTOSE = NO ENZYMES needed.

To conserve energy the cell turns off the production of these proteins, as follows: When the lactose is removed from the repressor, the repressor returns to its original shape and binds to the operator. Step 5: Place the REPRESSOR over the operator. See figure 8.
repressor This 2" clear tubing is slit down its length and is placed onto the operator.

Step 6: Slide the RNA polymerase from left to right across the operon, toward the operator. When RNA polymerase hits the repressor, it is blocked and can go no farther. Since RNA polymerase is blocked, it cannot transcribe the lac Z, lac Y, and lac A genes. The operon is now turned off. PART C: Turned on, lactose is back!

(blue)

(green)

(red)

(yellow)

(orange)

(white)

regulator promotor operator

Lac Z

Lac Y

Lac A

Figure 8

What happens when lactose is once again present and the cell needs to utilize it for energy? How is the repressor removed? When lactose is again present, it acts as an inducer of the operon. It enters the cell and binds to the repressor. Step 7: Place the colored pieces of paper back under the operon. Step 8: Push the lactose (paperclip) through the slit and hole in the repressor and push the repressor off the operon. See figure 9. When lactose binds to the repressor, it causes a conformational change that allows the repressor to fall off the DNA operator sequence. The repressor is unable to bind to the operator. Step 9: Try to place the REPRESSOR over the operator without moving the lactose. With the lactose in place, the repressor cannot bind to the operator. Since the repressor is unable to bind to the operator, the RNA polymerase is free to move along the DNA so that transcription can once again occur.

regulator promotor operator

Lac Z

Lac Y

Lac A

Figure 9

Step 10: Slide the RNA polymerase onto the operon and move it from left to right over the operon. Slide the colored pieces of paper (enzymes) out from under the operon model and toward you. (This is a repeat of Part A.) p. S5
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QUESTIONS
1. What is an operon and where is it found?

2. Draw the lac-operon and its genes. Label the following structures: operator, regulator, structural genes, and promoter. Briefly describe the function(s) of each structure.

3. Draw an operon as it appears when it is turned on. Label all structures not labeled in question 2.

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4. Draw an operon as it appears when it is turned off. Label all structures not labeled in questions 2 and 3.

5. Explain how the lac operon is involved in the regulation of protein synthesis.

6. What is the biological significance of operons?

7. Define transcription and translation and explain their relationship to each other.

8. Lac I codes for the repressor protein. Describe the effect on a cells regulation of -galactosidase if lac I did not exist in that cell or became mutated and could not make a functional repressor?

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