4546-lacoperon insts

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LacOperon: Turning On Your

Genes Activity Kit 47384

KIT CONTENTS

Item Quantity/Volume Description1 1 box Colored metal paper clips2 3 feet Tubing, 1/43 3 feet Tubing, 5/16

4 1 roll Red electrical tape5 15 Dowels with labels

6 1 package Yellow paper7 1 package White paper

8 1 package Orange paper9 1 Instructional video10 1 Set of instructions (this booklet):

Teachers Guide: Pages 1 - 7 Student Guide (reproducible): Pages S1 S7

Additional materials needed:transparent tape

scissorspermanent markers

rulerstrays 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 notall expressed at the same time. Genes are turned on (transcribed and translated) andturned 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 earlydevelopment. 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 ofthe persons life.

An operon is a section of DNA responsible for turning on and turning off the genes atspecific 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 therepressor protein). Repressor proteins prevent transcription of mRNA by RNA polymerase

by binding to the operator.

The lacoperon is the operon that allows the body to metabolize and utilize lactose (ab-

sorbed from the intestinal environment) as a source of energy.

In the presence of lactose, the lacoperon is turned on and the LacZ, LacY, and LacAenzymes coded for by genes on the lac operon can be synthesized and the lactose can bemetabolized. 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 willthen use a simple model to illustrate how and when the lacoperon is turned on and off. A

worksheet is also included, to reinforce and test students understanding of concepts dis-cussed and demonstrated in the lab.

This kit includes enough materials for 15 student lab stations. Students may work individu-ally or in small groups.


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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 beassigned 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 lacoperon.

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 thetubing across from the slit, 1 from the end. Using a perma-nent marker, label each of these tubes with the word repres-sor.

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 aroundone end, so that it covers half of the tube. Do not covereither open end of the tube with the tape. Using a perma-

nent marker, label each of these tubes with the words RNApolymerase.

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.

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red tape

2" clear tubing withslit down its length.

Unfolded paper clip is pushedthrough the tube so it can't beapplied 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 lacpermease.

-galactosidase** (see ** note below)

From the yellow construction paper, cut 15 - 1 x 1 yellow squares. Label each square with theword -galactosidase.

Acetylase** (see ** note below)

From the white construction paper, cut 15 - 1 diameter circles. Label each circle with the wordacetylase.

** 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, andacetylase 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 (lacoperon)1 RNA POLYMERASE tube1 split REPRESSOR tube

1 -galactosidase yellow square

SUGGESTED LESSON PLAN

1. With students, review transcription, translation, gene expression, relevant vocabulary, and therole of the lacoperon in lactose metabolism. Background information is provided in the StudentGuide.

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 andso forth. (Refer to the list below.)

lacoperon labeled dowelpromoter green section of dowel

regulator blue section of doweloperator red section of dowel

RNA polymerase clear tubing with red taperepressor 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 stu-

dents repeat parts B and C of the lab several times.

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1 permease orange triangle.1 acetylase white square.

1 lactose/inducer paper clip (first bend straightened)

Transparent tape (not included in kit)


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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 LacOperon 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 lacoperon is off when both glucose and lactose are present together in the

environment.

3. Explain how -galactosidase in E. colidiffers 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 poly-

merase 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 LacOperon.

[Online]. Available http://www.science-projects.com/beta-galase.htm

Ingram, Vernon, MIT Biology Hypertext Book, The LacOperon. [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

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Schleicher, Phil, Thomas, Ron, transformation of E.Coli, Production and Assay of -galac-tosidase by an Aerobic Fermentation. [Online]. Available http://www.uwrf.edu/biotech/

workshop/activity/act14/act14.htm, Summer 1996.

Essandoh, Nelly. Transcriptional Control in Bacteria: LacOperon, Trp Operon, and catabo-lite repression. [Online]. Available http://www.dartmouth.edu/~cbbc/courses/bio23/bio23-

1998/lecture-notes/05-TranscContBacteria.html , July 9, 1998

McGill University, The LacOperon 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.. MenloPark, 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 operatorwhich is responsible for turning these genes on and off. Operons are locatedwithin the chromosomes of an organism.

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

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.

regulator

(green)

operator Lac Z

(orange)

Lac A

(yellow)

Lac Y

(white) (red)

promotor

(blue)

mRNA + ribosomes

galactosidase permease acteylase


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

question 2.

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 lacoperon is involved in the regulation of protein synthesis.

The lacoperon turns on the structural genes that direct the production of en-

zymes needed to metabolize lactose when it is present in a cells environment and

is needed for energy. The lacoperon also turns off those structural genes when

lactose is absent from the cells environment or is not needed for energy.

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regulator

(green)

operator Lac Z

(orange)

Lac A

(yellow)

Lac Y

(white) (red)

promotor

(blue)

mRNA + ribosomes

galactosidase permease acetylase

RNApolymerase

repressor

lactose

blocked

change

in shape

regulator

(green)

operator Lac Z

(orange)

Lac A

(yellow)

Lac Y

(white) (red)

promotor

(blue)

RNApolymerase

repressor

RNA polymerase binds butcannot move to transcribe

No mRNA and no protein (enzymes)


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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 comple-

mentary 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. LacI codes for the repressor protein. Describe the effect on a cells regulation of -galactosidase if lacI 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 couldnot 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 anunnecessary expenditure of the cells energy and resources.

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

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

www.sciencekit.com

Carol BullockScience TeacherDanbury High SchoolDanbury, TX


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LacOperon: How To Turn Genes On!

Student Guide

VOCABULARY

Operon section of DNA responsible for turning on and turning off specific genes at specific times. Anoperon contains structural genes (genes that code for polypeptides), a promoter (initiation site wheretranscription begins), and an operator (the binding site for the repressor protein).

Lacoperon(entire labeled dowel) operon that allows the body to metabolize and utilize lactose (ab-

sorbed 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 at-

taches.

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 RNApolymerase 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 tran-

scribes the DNA in the gene into molecules of mRNA. The mechanism for control of gene expression wasdescribed 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 requiredfor enzyme production is referred to as an operon.


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Figure 1

Figure 2

regulator

(green)

operator Lac Z

(orange)

Lac A

(yellow)

Lac Y

(white) (red)

promotor

(blue)

mRNA + ribosomes

galactosidase permease acteylase

H O

2

Lactose Water Galactose Glucose

C H O H O C H O C H O12 22 11 2 6 6 12612 6

HHO

HOH

O

C

H

HH O

2

H

HO

H

OHHO

HH

HO

HOH

O

C

H

HH O

2

HHO

HH

HO

HOH

O

C

H

HO

HH O

2

HHO

HOH

O

C

H

HH O

2

HO

H

HO

H

Lactase

When E. coliis in an environment that contains lactose, a group of genes found on the lac operon allow thebacterium 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 foron 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 ofacetylase (the product of lac A) is unclear.

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 RNApolymerase 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 bemetabolized. See figure 3.


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regulator

(green)

operator Lac Z

(orange)

Lac A

(yellow)

Lac Y

(white) (red)

promotor

(blue)

RNApolymerase

repressor

RNA polymerase binds butcannot move to transcribe

No mRNA and no protein (enzymes)

regulator

(green)

operator Lac Z

(orange)

Lac A

(yellow)

Lac Y

(white) (red)

promotor

(blue)

mRNA + ribosomes

galactosidase permease acetylase

RNApolymerase

repressor

lactose

blocked

change

in shape

Figure 3

Figure 4

When lactose is removed, the repressor returns to its original shape and binds to the DNA, so that RNA poly-

merase can no longer get past the promoter. No RNA and no proteins are made. See 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 lacoperon.


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1 permease orange triangle*

1 acetylase white square* 1 lactose / inducer paper clip (with first bend straightened)

Transparent tape

Lac Z

(yellow) (orange)

Lac Y Lac A

(white)

operator

(red) (green)

promotorregulator

(blue)

RNA polymerase

Lac Z

(yellow) (orange)

Lac Y Lac A

(white)

operator

(red) (green)

promotorregulator

(blue)

Figure 5

Figure 6

RNA polymerase

yellow orange red

tape togetherusing clear tape

Lac Z

(yellow) (orange)

Lac Y Lac A

(white)

operator

(red) (green)

promotorregulator

(blue)

Figure 7

PROCEDURE

Before starting this lab, make sure you have all of the following materials.

1 labeled dowel (lacoperon)

1 RNA POLYMERASE tube 1 split REPRESSOR tube

1 -galactosidase yellow square*

*NOTE: If these items are not included, prepare the permease, -galactosidase and acetylase shapes, asdirected 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 lacoperon must be

turned on in order to produce the enzymes needed to metabolize the lactose. How is this done? Simulate theprocess as follows:

Step 1: Lay the operon on the tablewith the REGULATOR (blue end) to

your left (see Figure 5)

Place each paper shape under itscorresponding (same color) lacgeneon the operon. For example, place the

yellow square under the yellow lacZgene.

Step 2: Slide the RNA polymerase over the left end of the operon. Slide the RNA polymerase along the DNAoperon model from left to right.

See figure 6.This action simulates

the transcription ofthe DNA. The lacZ,lacY, and lacA

genes will be tran-scribed and trans-

lated and theirrespective enzymes

(-galactosidase,permease, andacetylase) will be

made.

Step 3: After RNApolymerase has

transcribed theentire operon, slide

the colored piecesof paper out fromunder the operon

model and towardyou. See figure 7.

At this point,lactose can bemetabolized into

galactose andglucose.


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repressor

This 2" clear tubing is slitdown its length and is placedonto the operator.

Lac Z

(yellow) (orange)

Lac Y Lac A

(white)

operator

(red) (green)

promotorregulator

(blue)

Figure 8

Lac Z Lac Y Lac Aoperatorpromotorregulator

Figure 9

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 Agenes do not need to be transcribed because -galac-tosidase, 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 theoperator.

Step 5: Place the REPRESSOR over theoperator. See figure 8.

Step 6: Slide the RNA polymerase fromleft to right across the operon, toward the

operator. When RNA polymerase hits therepressor, it is blocked and can go nofarther.

Since RNA polymerase is blocked, it

cannot transcribe the lac Z, lac Y, and lacAgenes. The operon is now turned off.

PART C: Turned on, lactose is back!

What happens when lactose is once again present and the cell needs to utilize it for energy? How is the repres-sor 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 theoperon. See figure 9.

When lactose binds to the repressor, it causes a

conformational change that allows the repressor tofall off the DNA operator sequence. The repressoris unable to bind to the operator.

Step 9: Try to place the REPRESSOR over the

operator without moving the lactose. With thelactose in place, the repressor cannot bind to the

operator.

Since the repressor is unable to bind to the opera-tor, the RNA polymerase is free to move along theDNA so that transcription can once again occur.

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 fromunder the operon model and toward you. (This is a repeat of Part A.)


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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, andpromoter. 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 lacoperon 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. LacI codes for the repressor protein. Describe the effect on a cells regulation of -galactosidase if lacI did

not exist in that cell or became mutated and could not make a functional repressor?

4546-lacoperon insts

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