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Glowingecoli

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Producing a Strain of E. coli that Glows in the Dark
Pamela C. Edgerton, Governor’s School for Government and International Studies,
Richmond, VA
INTRODUCTION
Description
In this exercise, students will create a luminescent population of bacteria by introducing into
Escherichia coli (E. coli) a plasmid that contains the lux operon. This operon is found in the
luminescent bacterium Vibrio fischeri and contains two genes that code for luciferase (the enzyme
that catalyzes the light-emitting reaction) and several genes that code for enzymes that produce the
luciferins (the substrates for the light-emitting reaction). The success of the transformation is readily
apparent, since E. coli colonies that take up this plasmid glow in the dark. In another group, a control
plasmid (pUC18) that does not contain the lux operon will be introduced into E. coli, and these cells
will not glow in the dark. Both plasmids contain an ampicillin-resistant gene, and therefore both cell
types will grow in the presence of the antibiotic. Each resistant colony growing on ampicillin-
nutrient agar plates represents a single transformation event, and only those colonies that have taken
up the plasmids will grow on the agar. The bioluminescent colonies represent bacteria that can
successfully express a metabolic marker.
Student Audience
This exercise is suitable for middle and high school students who have discussed concepts related to
DNA structure and function, the tools and methods of biotechnology, and present-day applications of
those tools and methods.
Goals for the Activity
The main goals for this activity are to

enable students to observe the experimental process called bacterial transformation, demonstrate the relationship between the genetic constitution of an organism and its physicalattributes, enable students to observe the change in phenotype caused by the uptake and expression of aknown plasmid sequence, and reinforce the need for sterile technique when working with bacteria.
Recommended Placement in the Curriculum
This exercise is best used in conjunction with a unit presenting information on the molecular basis of
heredity, gene expression, the genetics of viruses and bacteria, and DNA technologies.
Developed through the National Science Foundation-fundedPartnership for the Advancement of Chemical Technology (PACT) STUDENT HANDOUT
Producing a Strain of E. coli that Glows in the Dark

Purpose
The purpose of this exercise is to demonstrate phenotype changes in bacteria that have been
transformed with an antibiotic-resistance gene and a metabolic marker and to introduce the concept
of recombinant DNA and cloning vectors.
Discussion
Plasmids are small, circular DNA molecules that exist apart from the chromosomes in most bacterial
species. Under normal circumstances, plasmids are not essential for survival of the host bacteria.
However, many plasmids contain genes that enable bacteria to survive and prosper in specific
environments. For example, some plasmids carry one or more genes that confer resistance to
antibiotics. A bacterial cell containing such a plasmid can live and multiply in the presence of the
drug. Indeed, antibiotic-resistant Escherichia coli (E. coli) isolated in many parts of the world
contain plasmids that carry the genetic information for protein products that interfere with the action
of many different antibiotics. In this laboratory, you will introduce a plasmid that contains an
ampicillin-resistance gene into E. coli.
One plasmid that you will use is called pUC18. Plasmid pUC18 contains only 2,686 nucleotide pairs(molecular weight = 2 x 106). The small size of this plasmid makes it less susceptible to physicaldamage during handling. In addition, smaller plasmids generally replicate more efficiently inbacteria and produce larger numbers of plasmids per cell. As many as 500 copies of this plasmidmay be present in a single E. coli cell. Plasmid pUC18 contains an ampicillin-resistance gene thatenables E. coli to grow in the presence of the antibiotic. Bacteria lacking this plasmid, or bacteriathat lose the plasmid, generally will not grow in the presence of ampicillin. The ampicillin-resistancegene of pUC18 codes for the enzyme beta-lactamase (penicillinase), which inactivates ampicillinand other penicillins.
In the laboratory, plasmids can be introduced into living bacterial cells by a process known astransformation. When bacteria are placed in a solution of calcium chloride (CaCl ), they acquire the ability to take in plasmid DNA molecules. This procedure provides a means for preparing largeamounts of specific plasmid DNA, since one transformed cell gives rise to clones that contain exactreplicas of the parent plasmid DNA molecule. Following growth of the bacteria in the presence ofthe antibiotic, the plasmid DNA can be readily isolated from the bacterial culture.
Plasmids are very useful tools for the molecular biologist because they serve as gene-carriermolecules. A basic procedure of recombinant DNA technology consists of joining a gene of interestto plasmid DNA to form a hybrid, or recombinant, molecule that is able to replicate in bacteria. Inorder to prepare a recombinant molecule, the plasmid and gene of interest are cut at precisemolecules and spliced together. After the hybrid plasmid molecule has been prepared, it is introducedinto E. coli cells by transformation. The hybrid plasmid replicates in the dividing bacterial cells toproduce an enormous number of copies of the original gene. At the end of the growth period, thehybrid molecules are purified from the bacteria, and the original gene of interest is recovered. Thismethod has enabled scientists to obtain large quantities of more than 1,000 specific genes, includingthe genes for human interferon, insulin, and growth hormone.
Developed through the National Science Foundation-fundedPartnership for the Advancement of Chemical Technology (PACT) Collection of Laboratory Activities: Producing a Strain of E. coli that Glows in the Dark DNA technology has triggered advances in almost all fields of biology by enabling biologists totackle specific questions with finer tools. By far the most ambitious research project made possible isthe Human Genome Project, a multibillion-dollar effort to determine the nucleotide sequence of theentire human genome. The entire human genome will be characterized by cloning in specializedvectors and by analyzing the clones to determine where their inserted DNA molecules reside on the23 chromosomes of the human genome. Such an analysis, aided by research on smaller genomes,produces a physical map of all the genes in the human genome. This map will be pivotal in researchconcerning the diagnosis of human genetic disorders and other diseases, the application of genetherapy, and the development of vaccines and other pharmaceutical products.
DNA technology also offers applications to the study of forensic science, problems relating to theenvironment, and the ever-growing need for better, more productive agriculture. DNA testing hasrefined the process of forensic analysis because not as much tissue is required and guilty individualscan be identified with a higher degree of certainty. With recent advances in restriction fragmentlength polymorphism (RFLP) analysis, polymerase chain reaction (PCR), and variable number oftandem repeats (VNTRs), the probability that two people will have exactly the same DNAfingerprints is very small. (For legal cases the probability is between one chance in 100,000 and onein a billion, depending on the number of genomic sites compared.) Environmental research has seen an increase in the applications of genetics engineering.
Microorganisms are being used to extract heavy metals from mining waste; clean up the environment;provide another source for metals such as lead, copper, and nickel; recycle wastes; and detoxify toxicchemicals. Sewage treatment facilities rely on the ability of microbes to degrade many organiccompounds into nontoxic forms. Chlorinated hydrocarbons are a real problem, and biotechnologistsare attempting to engineer microorganisms that can be incorporated into the treatment process todegrade these compounds. Eventually the microorganisms may be incorporated into themanufacturing of these toxic chemicals, thus preventing their release as waste.
Agricultural applications of DNA technology include research in the areas of animal husbandry andmanipulating plant genes. Products of recombinant DNA methods are already being used to treatfarm animals. These include new or redesigned vaccines, antibodies, and growth hormones. Bovinegrowth hormone (BGH), made by E. coli, increases milk production by approximately 10% in milkcows and improves weight gain in beef cattle. Transgenic organisms (organisms that contain genesfrom other species) have been developed for potential agricultural and medical uses (e.g., organtransplants, production of human hormones, etc.). Genetic engineers working with plant genescommonly use DNA vectors to move genes from one organism to another. Several companies havedeveloped strains of wheat, cotton, and soybeans carrying a bacterial gene that makes the plantsresistant to herbicides used by farmers to control weeds. Tomatoes containing genes that retardspoilage have been approved by the Food and Drug Administration (FDA). A number of crop plantshave been engineered to be resistant to infectious pathogens and pest insects. Increasing the abilityof bacteria to “fix” nitrogen or transferring the ability to fix nitrogen directly to plants would reduceor eliminate the need for expensive and environmentally detrimental fertilizers.
DNA technologies have many uses in fields as diverse as medicine, agriculture, forensic science,ecology, and industry. The early successes in research point to a revolution just around the corner.
Using these technologies wisely will become a challenge of the next century.
(Excerpts taken from Campbell, 1996, Ogden and Adams, 1989, Micklos and Bloom, 1989, andAnderson, 1995.) Developed through the National Science Foundation-fundedPartnership for the Advancement of Chemical Technology (PACT) Collection of Laboratory Activities: Producing a Strain of E. coli that Glows in the Dark Materials
Per class

20 petri dishes containing ampicillin-nutrient agar 10% bleach solution, soapy water, or disinfectant (such as Lysol®) Safety, Handling, and Disposal
It is your responsibility to specifically follow your institution’s standard operating procedures
(SOPs) and all local, state, and national guidelines on safe handling and storage of all chemicals and
equipment you may use in this activity. This includes determining and using the appropriate personal
protective equipment (e.g., goggles, gloves, apron). If you are at any time unsure about an SOP or
other regulation, check with your instructor. Dispose of used reagents according to local ordinances.
When dealing with biological materials, take particular precautions as called for by the kitmanufacturer or supplier. A commensal organism of Homo sapiens, E. coli is a normal part of thebacterial fauna of the human gut. It is not considered pathogenic and is rarely associated with anyillness in healthy individuals. Adherence to simple guidelines for handling and disposal makes workwith E. coli a nonthreatening experience for instructor and students.
Wear gloves when handling bacteria. When pipetting a suspension culture, keep your nose andmouth away from the tip end to avoid inhaling any aerosol that might be created.
Avoid overincubating plates. Because a large number of cells are inoculated, E. coli is generally theonly organism that will appear on plates incubated for 12–24 hours at 37°C. However, with longerincubation, contaminating bacteria and slower-growing fungi may arise. If students will not be ableto observe plates following initial incubation, refrigerate plates to retard growth of contaminants.
Always reflame the inoculating loop or cell spreader one final time before setting it down on the labbench. Wipe down lab benches with 10% bleach solution, soapy water, or disinfectant (such asLysol) at the end of the activity. Wash your hands before leaving the laboratory.
Collect bacterial cultures, as well as tubes, pipets, and transfer loops that have come into contactwith the cultures. Disinfect these materials as soon as possible after use. Contaminants, often smellyand sometimes potentially pathogenic, can be cultured over a period of several days at roomtemperature. Disinfect bacteria-contaminated materials in one of two ways: Developed through the National Science Foundation-fundedPartnership for the Advancement of Chemical Technology (PACT) Collection of Laboratory Activities: Producing a Strain of E. coli that Glows in the Dark Treat with 10% bleach solution (5,000 ppm available chlorine). Immerse contaminated pipets,transfer loops, and open tubes directly in a sink or tub containing bleach solution. Plates shouldbe placed in a sink or tub and flooded with bleach solution. Let materials stand in bleach solutionfor 15 minutes or more. Then drain excess bleach solution, seal the materials in a plastic bag, anddispose of them as regular garbage.
Autoclave these items at 121°C for 15 minutes. Tape three or four culture plates together andtightly close tube caps before disposal. Collect contaminated materials in an autoclavable bagand seal the bag before autoclaving. Dispose of the materials as regular garbage.
Procedure
In this exercise, plasmid lux and a control plasmid (pUC18) will be introduced into E. coli by
transformation. There are four basic steps to the procedure:
A. Treat bacterial cells with CaCl solution in order to enhance the uptake of plasmid DNA. Such CaCl -treated cells are said to be “competent.” (This step should be performed by the instructor before or during the laboratory session.) B. Incubate the competent cells with plasmid DNA.
C. Select those cells that have taken up the plasmid DNA by growth on an ampicillin-containing D. Examine the cultures in the dark.
A. Preparation of competent cells(These steps should be performed by the instructor.)1. Place a vial of CaCl solution and the tube of E. coli in the ice bath.
2. Using a sterile pipet, transfer about 0.5 mL CaCl solution to the tube containing the bacteria.
3. Using the same pipet, transfer the contents of this tube back into the vial that contains most of 4. Tap the vial with the tip of your index finger to mix the solution.
5. Incubate the cells for about 10 minutes on ice. The cells are then called competent because they can take up DNA from the medium. If desired, the cells can be stored in the CaCl solution for B. Uptake of DNA by competent cells1. Label one small tube “C DNA” (for control DNA) and one tube “L DNA” (for plasmid lux).
2. Place the two tubes in an ice bath.
3. Using a sterile micropipet, add 10 µL control plasmid to the tube labeled “C DNA” and 10 µL plasmid lux to the tube labeled “L DNA.” Developed through the National Science Foundation-fundedPartnership for the Advancement of Chemical Technology (PACT) Collection of Laboratory Activities: Producing a Strain of E. coli that Glows in the Dark 4. Gently tap the tube of competent cells with the tip of your index finger to ensure that the cells are in suspension. Then, using a sterile transfer pipet, add 6 drops of the competent cells(approximately 120 µL) to each of the two tubes. Tap each of these tubes with the tip of yourindex finger to mix these solutions, and store both tubes on ice for 10–15 minutes. Thecompetent cells, which are suspended in the CaCl solution, will now begin to take up the plasmid DNA. During this time, one member of the class should obtain two additional tubes.
Add 6 drops of competent cells to each tube and label the tubes “NP” (no plasmid).
5. Transfer the tubes to a water bath preheated to 37°C, and allow them to sit in the bath for 5 6. Use a sterile pipet to add about 0.7 mL nutrient broth to each tube and incubate the tubes at 37°C for 30–45 minutes. This incubation period allows the bacteria time to recover from the CaCl2treatment and to begin to express the ampicillin-resistant gene on the plasmid.
C. Selection of cells that have taken up the plasmid by growth on an ampicillin- 1. Obtain four ampicillin-nutrient agar plates from your instructor. Label one plate “C DNA,” another plate “L DNA,” and the remaining plates “NP.” 2. Using a sterile pipet, remove 0.25 mL mixed bacterial suspension from the “C DNA” tube, remove the lid from the “C DNA” plate, and dispense the bacteria onto the agar. Use aninoculating loop to spread the bacteria evenly onto the agar surface.
3. Transfer 0.25 mL bacterial suspension from the “L DNA” tube to the “L DNA” plate and spread these cells onto the agar surface as described in the previous step.
4. Cells from the two tubes that did not contain plasmids (NP) should be plated onto two plates 5. Replace the lids on the plates, and leave the plates at room temperature until the liquid has been 6. Invert the plates and incubate them in the light-free room at room temperature.
Data Analysis and Study Questions
1. Predict the growth on the following plates by marking “+” for growth and “–” for no growth in
Developed through the National Science Foundation-fundedPartnership for the Advancement of Chemical Technology (PACT) Collection of Laboratory Activities: Producing a Strain of E. coli that Glows in the Dark 2. Colonies should appear in about 2–3 days at room temperature. Plates must be viewed at that time, since bioluminescence decreases with time after colony formation. Allow at least 3 minutesfor the eyes to adjust to the dark in a light-free room. View your plates and the plates of yourclassmates in the dark and then in the light. Record your results in the following table. Were theresults as expected? Explain possible reasons for variations from expected results.
3. Why do the cells transformed with pUC18 and plasmid lux grow in the presence of ampicillin? 4. Name one enzyme that is produced by cells transformed with plasmid lux that is not produced by 5. Remembering that plasmid size will affect the efficiency of transformation, which plate would be Suggested Readings
Access Excellence®. About Biotech. www.accessexcellence.org//AB/index.html (accessed April 4,
National Human Genome Research Institute Glossary of Genetic Terms. www.nhgri.nih.gov/DIR/ VIP/Glossary/pub_glossary.cgi (accessed April 4,2000).
The University of Arizona. The Biology Project. www.biology.arizona.edu/ (accessed April 4, 2000).
References
Anderson, J. “Producing a Strain of E. coli that Glows in the Dark,” Modern Biology. 1995.
Campbell, N.A. Biology; Benjamin/Cummings: Menlo Park, CA, 1996.
Micklos, D.A.; Bloom, M.J. DNA Science: A First Laboratory Course in Recombinant DNA
Technology; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1989.
Ogden, R.; Adams, D.A. “Recombinant DNA Technology: Basic Techniques,” Carolina Tips. 1989, Ogden, R.; Adams, D.A. “Recombinant DNA Technology: Applications,” Carolina Tips. 1989, 52, Developed through the National Science Foundation-fundedPartnership for the Advancement of Chemical Technology (PACT) INSTRUCTOR NOTES
Producing a Strain of E. coli that Glows in the Dark

Time Required
The activity may be conducted using the following time table:
Group Size
The activity may be conducted in groups of two or three students.
Materials
If the author’s instructions call for use of a commercial kit and specific materials provided in that kit,
we strongly recommend that you use the recommended materials to attain the desired results. When
using a commercial kit, read and follow the instructions provided by the kit manufacturer. The
author’s procedure provided in this activity is not necessarily intended to duplicate or reproduce the
manufacturer’s instructions. Rather, the procedure has been provided by the author as a summary of
the general steps to follow.

This lab kit can be purchased from Modern Biology, Inc., 111 North 500 West; West Lafayette,Indiana 47906. 1-800-733-6544, FAX 1-765-743-7612. 20 Petri dishes containing ampicillin-nutrient agar 10% bleach solution, soapy water, or disinfectant (such as Lysol®) Developed through the National Science Foundation-fundedPartnership for the Advancement of Chemical Technology (PACT) Collection of Laboratory Activities: Producing a Strain of E. coli that Glows in the Dark Safety, Handling, and Disposal
As the instructor, you are expected to provide students with access to SOPs, MSDSs, and other
resources they need to safely work in the laboratory while meeting all regulatory requirements.
Before doing this activity or activities from other sources, you should regularly review special
handling issues with students, allow time for questions, and then assess student understanding of
these issues.
When dealing with biological materials, take particular precautions as called for by the kitmanufacturer or supplier. A commensal organism of Homo sapiens, Escherichia coli (E. coli) is anormal part of the bacterial fauna of the human gut. It is not considered pathogenic and is rarelyassociated with any illness in healthy individuals. Adherence to simple guidelines for handling anddisposal makes work with E. coli a nonthreatening experience for instructor and students.
Wear gloves when handling bacteria. When pipetting a suspension culture, keep your nose andmouth away from the tip end to avoid inhaling any aerosol that might be created.
Avoid overincubating plates. Because a large number of cells are inoculated, E. coli is generally theonly organism that will appear on plates incubated for 12–24 hours at 37°C. However, with longerincubation, contaminating bacteria and slower-growing fungi may arise. If students will not be ableto observe plates following initial incubation, refrigerate plates to retard growth of contaminants.
Always reflame the inoculating loop or cell spreader one final time before setting it down on the labbench. Wipe down lab benches with 10% bleach solution, soapy water, or disinfectant (such asLysol) at the end of the activity. Wash your hands before leaving the laboratory.
Collect bacterial cultures, as well as tubes, pipets, and transfer loops that have come into contactwith the cultures. Disinfect these materials as soon as possible after use. Contaminants, often smellyand sometimes potentially pathogenic, can be cultured over a period of several days at roomtemperature. Disinfect bacteria-contaminated materials in one of two ways: Treat with 10% bleach solution (5,000 ppm available chlorine). Immerse contaminated pipets,transfer loops, and open tubes directly in a sink or tub containing bleach solution. Plates shouldbe placed in a sink or tub and flooded with bleach solution. Let materials stand in bleach solutionfor 15 minutes or more. Then drain excess bleach solution, seal the materials in a plastic bag, anddispose of them as regular garbage.
Autoclave these items at 121°C for 15 minutes. Tape three or four culture plates together andtightly close tube caps before disposal. Collect contaminated materials in an autoclavable bagand seal the bag before autoclaving. Dispose of the materials as regular garbage.
Dispose of used reagents according to local ordinances.
Developed through the National Science Foundation-fundedPartnership for the Advancement of Chemical Technology (PACT) Collection of Laboratory Activities: Producing a Strain of E. coli that Glows in the Dark Points to Cover in the Pre-Lab Discussion
Discuss the concept of bacterial transformation:

incubation of cells and uptake of plasmid DNA; selection of cells using an ampicillin-containing medium; contamination prevention through use of sterile techniques.
Stress the importance of proper disposal of used equipment and materials to prevent the spread ofbacteria. Review the proper use of a micropipet. Note that an inoculating loop can be used todispense 10 µL (10 µL = one loop full).
Procedural Tips and Suggestions
A room with lights out and shades drawn does not provide enough darkness to observe
luminescence. The room must be in total darkness to the point of not seeing your hand in front of
your face.
Luminescence is apparent at 18–24 hours after transformation when plates are incubated at 37°C orat 2–3 days at room temperature. After these times, the amount of light emitted decreases.
Luminescence can be maintained if the cells that carry the lux plasmid are subcultured every 3–4days on agar-ampicillin plates.
Instead of using the microliter pipets, the inoculating loops can be used to dispense 10 µL (10 µL =one loop full).
During the uptake of DNA by competent cells, have an activity to occupy your students. The 30–45minutes it takes for the cells to incubate can be a good time to discuss the process of bacterialtransformation.
Preparation of competent cells by the instructor can save time in the lab session.
Plausible Answers to Questions
1. Predict the growth on the following plates by marking “+” for growth and “–” for no growth in
Developed through the National Science Foundation-fundedPartnership for the Advancement of Chemical Technology (PACT) Collection of Laboratory Activities: Producing a Strain of E. coli that Glows in the Dark 2. Colonies should appear in about 2–3 days at room temperature. Plates must be viewed at that time, since bioluminescence decreases with time after colony formation. Allow at least 3 minutesfor the eyes to adjust to the dark in a light-free room. View your plates and the plates of yourclassmates in the dark and then in the light. Record your results in the following table. Were theresults as expected? Explain possible reasons for variations from expected results.
If bioluminescent colonies are growing, then students can just answer “yes.” If nobioluminescent colonies appear to be growing on the plates, then the students should answer“no” and provide possible explanations for the results. The following might be an acceptableexplanation: There were no bioluminescent colonies growing on the L DNA plate. The bacterialcells might not have been competent to uptake the plasmid. There could have been contaminationdue to not using sterile technique. The plasmid might not have been of the proper concentration. 3. Why do the cells transformed with pUC18 and plasmid lux grow in the presence of ampicillin? Both plasmids grow because they contain an ampicillin-resistance gene that codes for a product(beta-lactamase) that breaks down ampicillin. 4. Name one enzyme that is produced by cells transformed with plasmid lux that is not produced by the cells transformed by pUC18.
Luciferase. 5. Remembering that plasmid size will affect the efficiency of transformation, which plate would be expected to show the fewest colonies?The plasmid lux plate would be expected to show the fewest colonies because that plasmid islarger than plasmid pUC18 and would be more difficult for the cells to absorb. The largerplasmid lux is also more susceptible to physical damage and probably replicates less efficiently. Extensions
This lab could be performed using a variety of different plasmids that are on the market today. Many
biological supply companies sell a host of recombinant DNA plasmids that would be suitable to
demonstrate bacterial transformation.
Extensions of this lab could include the following: Analyze the effects of growth temperature and time on the glowing process. This analysis can becarried out by replating the transformed cells onto fresh nutrient-ampicillin agar plates.
Isolate plasmid lux from transformed cells using plasmid DNA isolation techniques. Theplasmids can then be digested with various restriction endonucleases to identify the plasmids thatwere cloned.
Developed through the National Science Foundation-fundedPartnership for the Advancement of Chemical Technology (PACT) Collection of Laboratory Activities: Producing a Strain of E. coli that Glows in the Dark References
Anderson, J. “Producing a Strain of E. coli that Glows in the Dark,” Modern Biology. 1995.
Campbell, N.A. Biology; Benjamin/Cummings: Menlo Park, CA, 1996.
Micklos, D.A.; Bloom, M.J. DNA Science: A First Laboratory Course in Recombinant DNA
Technology; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY, 1989.
Ogden, R.; Adams, D.A. “Recombinant DNA Technology: Basic Techniques,” Carolina Tips. 1989, Ogden, R.; Adams, D.A. “Recombinant DNA Technology: Applications,” Carolina Tips. 1989, 52, Developed through the National Science Foundation-fundedPartnership for the Advancement of Chemical Technology (PACT)

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