Antibiotics, Magic Pill or Overkill?
Easy PCR
Student Materials
Introduction…………………………………………………………………………………………………………………………. 2
Pre-Lab Questions………………………………………………………………………………………………………………… 6
Lab Protocol…………………………………………………………………………………………………………………………. 7
Data Collection Worksheet…………………………………………………………………………………………………. 10
Post-Lab Questions and Analysis………………………………………………………………………………………… 11
Last updated: 3/15/2017
Antibiotics, Magic Pill or Overkill?
Introduction
Antibiotics are powerful medicines that fight bacterial infections. There is no doubt that antibiotics save
lives. Before 1945 and the routine use of antibiotics, cuts, scrapes, childbirth, strep throat, surgery and
pneumonia often proved fatal. An antibiotic is any small molecule that inhibits the growth of
microorganisms like bacteria or fungi (Figure 1). Antibiotics are naturally produced by many
microorganisms, most often by other soil bacteria and fungi.
Figure 1. Microorganisms (a) Typical bacteria cell. (b) Typical fungal hyphae cells.
(a)
(b)
(http://sciencewithsteve.co.nz/)
But wait…why would microorganisms in the soil produce antibiotics? Wouldn’t producing antibiotics kill
the bacteria producing them?
Remember that soil microorganisms are decomposers and in any small patch of soil there are hundreds
or thousands of different microorganisms competing with each other for dead plant material that they
use for food. Different microorganisms produce different antibiotics. Each kind of antibiotic-producing
bacteria or fungi has evolved to make an antibiotic that can inhibit the growth of competing organisms
without inhibiting itself. Any soil bacteria or fungi that can limit the growth of different nearby species
will have more resources and therefore will have an evolutionary advantage.
The production of antibiotics by microorganisms may explain why a mash
of moldy grain was used to treat skin infections in ancient and rural
cultures (Moulds in ancient and more recent medicine, Mycologist:
3(1):21-23, January 1989). The discovery of penicillin, the first modern
antibiotic, has been attributed to Alexander Fleming (Figure 2). As the story
goes, one day in 1928, Alexander Fleming returned from a vacation to his
laboratory. While sorting petri dishes containing colonies of bacteria he
noticed something unusual. On one petri plate there was a large mass of
mold and, although there were many bacterial colonies on the plate, no
colonies were present near the mold. Fleming concluded that the mold was
producing something that inhibited bacterial growth. In fact, Fleming had
discovered a form of penicillin that inhibits bacterial cell wall synthesis and
thus kills newly made bacterial cells.
Figure 2. Alexander Fleming at
work in his laboratory.
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It took many scientists and many years to move from Fleming’s observation to the production of
penicillin for use with patients. Along the way, many other antibiotics like streptomycin,
chloramphenicol and tetracycline were discovered in soil bacteria. In addition, scientists have made
small modifications to naturally occurring molecules to produce man-made antibiotics. For example, the
man-made antibiotic ampicillin is just like penicillin except it has an added amino group. Both drugs
inhibit bacterial cell wall synthesis, however, the extra amino group on ampicillin makes it more
effective than penicillin against some Gram-negative bacteria.
To complicate matters, evolutionary changes in one organism can affect the evolution of interacting
organisms. In this case, co-evolution has resulted in bacteria and fungi that are resistant to the
antibiotics released by competing microorganisms. The resistant organisms have evolved genes that
encode proteins that prevent the antibiotic from killing the cell. Some of the proteins can break down
the antibiotic before it can harm the cell, some can prevent antibiotics from entering the cell, and others
allow the cell to pump out the antibiotic before it can do harm. For example, the ampicillin resistance
gene, ampR, encodes a protein that breaks ampicillin down before it can interfere with cell wall synthesis
and kill the cell.
In bacterial cells, the gene or genes that protect against antibiotics are carried on small, circular, doublestranded DNA molecules called plasmids (Figure 3). Cells can pick plasmids up from the environment
and plasmids can be passed between living bacterial cells. Cells that carry a plasmid with the ampR gene
will not be killed by ampicillin.
Widespread overuse of many different antibiotics has resulted in a dramatic increase in antibiotic
resistant bacteria. Antibiotic resistance in the types of bacteria that cause human disease is increasing in
the United States and worldwide. In these cases, antibiotics can no longer be used to treat infections.
Antibiotic resistance has resulted in an increase in serious health problems, in death due to infections,
and in the cost of health care.
In this lab you will use the Polymerase Chain Reaction (PCR) to test different bacterial strains to
determine if they are antibiotic resistant. You will be given DNA from bacteria known to be antibiotic
resistant and DNA from an unknown bacterial strain. This DNA will be the template for PCR primers
designed to specifically amplify only the ampR gene (Figure 3). If the sample has the ampR gene, PCR
should result in many copies of a DNA fragment of about 700 base pairs. If the sample does not have the
ampR DNA, the PCR reaction will not generate any DNA fragments.
Figure 3. Ampicillin resistant gene on a plasmid in a bacterial cell.
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Let’s look at how PCR works using the diagrams below.
First. The template DNA must be separated into single-stranded DNA molecules by increasing the
temperature to 94 °C. The high temperature disrupts the hydrogen bonds between the complementary
base pairs.
94 °C
Second. The reaction temperature is decreased to a temperature between 50 and 70 °C. This temperature,
called the annealing temperature, is determined by the sequence of the primers and is set to optimize
primers binding (annealing) to the complementary sequences on the template DNA. Because primers will be
incorporated into each new molecule, the primers are present in vast excess. The high concentration also
increases the frequency of primers binding to the template.
50-70 °C
Third. The reaction temperature is increased to the optimal temperature for the DNA polymerase being
used in the reaction. There are several thermostable DNA polymerases used in PCR and each has a
slightly different optimal temperature. At this temperature, the DNA polymerase adds nucleotides to
the 3’ end of the primers in a manner that is complementary to the template strand. In a reaction
mixture, there is an abundance of polymerase so all the template strands are copied simultaneously.
68 °C
Polymerase Chain Reaction Graphics from https://commons.wikimedia.org/wiki/File:PCR_Steps.JPG
These three steps constitute one PCR cycle. A typical PCR amplification repeats the three step cycle 2030 times. The target DNA (the DNA flanked by the primers) is doubled during each cycle. Beginning with
a single DNA molecule, 20 cycles can produce over a million copies.
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The following schematic shows the first four cycles of PCR amplification. Notice that as the number of
cycles increases, the percentage of PCR products of the desired length also increases.
Figure 4. Schematic of 4 cycles of PCR.
Polymerase Chain Reaction image from https://commons.wikimedia.org/wiki/File:PCR_basic_principle1.jpg
The presence or absence of the PCR amplified DNA fragments will be determined by agarose gel
electrophoresis. If needed, please review the document ”A Guide to Agarose Gel Electrophoresis” for
background on the technique and applications of agarose gel electrophoresis.
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Antibiotics, Magic Pill or Overkill?
Pre-Lab Questions
Directions: After reading through the introduction and protocol for the Antibiotics, Magic Pill or
Overkill lab, answer the questions below.
1. What is an antibiotic? Where are antibiotics found in nature?
2. What does the term antibiotic resistance mean as applied to bacterial cells?
3. List the essential components required for PCR.
4. List the three steps of a PCR cycle and describe what occurs at each step.
5. Briefly describe how agarose gel electrophoresis separates molecules of different size?
6. You find that bacterial cells containing a specific plasmid live and reproduce in the presence of the
antibiotic tetracycline, but bacterial cells without the plasmid are killed in the presence of tetracycline.
What gene must be carried on this specific plasmid that allows cells to live and reproduce in the
presence of the tetracycline? Propose a possible mechanism for the protein encoded by this gene that
allows cells to live and reproduce in the presence of the tetracycline?
7. Say that your dog has a bacterial skin infection. Explain why it would be beneficial to know whether or
not the bacteria causing this infection are resistant to penicillin.
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Antibiotics, Magic Pill or Overkill?
Lab Protocol
In today’s lab, you will be analyzing the DNA from a strain of unknown bacteria to determine if it carries
the gene for ampicillin resistance. You will use PCR to amplify the ampicillin resistance (ampR) gene
from a strain known to be resistant to ampicillin (control) and from an unknown strain (test). The results
of the PCR reaction will be determined by agarose gel electrophoresis. Prior to today’s experiment, lab
technicians have collected DNA samples from the two strains of bacteria and ordered primers specific
for the ampR gene.
Materials:
Check your workstations to make sure all supplies are present before beginning the lab.
Student Workstation
Common Workstation
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1 p200 micropipette and tips (optional)
1 p20 micropipette and tips
1 microcentrifuge tube rack
1 extra fine point permanent marker
1 ice bucket with ice
3 small thin-walled PCR tubes
1 tube with 500 L nuclease-free water
1 tube Control template DNA with 5 L dilute plasmid
1 tube Test template DNA with 5 L dilute plasmid
1 tube Primer mix with 20 L forward and reverse primers
for the antibiotic resistance gene
1 tube OneTaq® with 85 L Quick-Load® 2X Master Mix
1 tube DNA Ladder with 12 L Quick Load ® Purple 100 bp
1 agarose gel (1.5%) with DNA stain
thermocycler
blue light or UV transilluminator
centrifuge (optional)
1X electrophoresis buffer
Caution: Keep template DNA, primers, OneTaq®, and your sample tubes on ice at all times.
Procedure:
1. Label two thin-walled PCR tube with your initials and C1 and C2 (for the controls). Label the other
PCR tube with your initials and T (for test).
2. Prepare the tubes as instructed in Table 1 that follows.
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Table 1. PCR Reaction Set-up.
Tube
Component
C1 (control 1)
C2 (control 2)
Test
Nuclease-free water
21 L
19 L
19 L
OneTaq® Quick-Load® 2X Master Mix
25 L
25 L
25 L
Primer Mix (1.5 M of each primer)
4 L
4 L
4 L
Template DNA (0.2 ng/l)
None
2 L (control DNA)
2 L (test DNA)
Reminder: Use a new pipette tip each time you transfer a solution.
3. If needed, briefly spin the tubes in the microcentrifuge to collect the contents at the bottom of the
tube. If you do not have a microcentrifuge, gently tap the tubes to collect the contents at the
bottom of the tube.
4. Place PCR tubes in PCR machine and run the following PCR program:
# of Cycles
1
20
1
Temperature
94 °C
94 °C
58 °C
68 °C
68 °C
Time
1 minute
15 seconds
15 seconds
30 seconds
2 minutes
5. When the PCR program is complete, remove your tubes from the PCR machine.
Tip: check the labels as you go and label again if needed!
Stopping Point: Check with your teacher before continuing with the protocol.
6. Obtain or pour a 1.5% agarose gel. This gel should include a DNA stain.
7. Using a p20, load 10 µL of 100 bp ladder to your gel.
8. Using a p20, load 6 µL of each of your samples onto the gel wells. Record the order that you loaded
your samples in Table 2 on the next page.
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Table 2. Order samples were loaded in gel.
Well
1
2
3
PCR
Sample
4
5
6
7
8
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Reminder: Use a new pipette tip for each sample.
9. Run the gels at constant voltage until the dye front is about 2/3 of the way down from the top of the
gel.
10. Turn off the power supply and examine your results. Be sure to take a photograph of your gel or
sketch your results in your lab notebook.
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Antibiotics, Magic Pill or Overkill?
Data Collection Worksheet
Directions: After completing the Antibiotics, Magic Pill or Overkill lab, answer the questions below.
1. On the image below draw what you see after gel electrophoresis
100 bp
Ladder
−electrode
1517
1200
1000
900
800
700
600
500
400
300
200
100
+electrode
2. Given the results from your gel, what can you conclude about the test bacteria?
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Antibiotics, Magic Pill or Overkill?
Post-Lab Questions and Analysis
Directions: After completing Antibiotics, Magic Pill or Overkill lab, answer the questions below.
1. What does any band on the gel represent?
2. Analyze your gel and answer the following questions.
• Do you see a band or bands in the control lanes?
• What was the purpose of the control 1 (C1)? Did you expect a band in the C1 lane?
•What was the purpose of the control 2 (C2)? Did you expect a band in the C2 lane?
3. Assume that you see a band in the C1 lane.
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Provide one scenario that would explain the presence of a C1 band.
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Give a statement about how the presence of a band in the C1 lane influences your
interpretation of the data from the test sample.
4. Assume that you do not see a band in the C2 lane.
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Provide one scenario that would explain the absence of a C2 band.
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Give a statement about how the absence of a band in the C2 lane influences your interpretation
of the data from the test sample.
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5. Do you see a band or bands in the test lane? Given your results from the control and the test lanes,
what can you conclude about the bacteria tested?
6. What gene were your primers designed to amplify?
7. Given your results, circle all of the petri plates on which the test bacteria will grow.
Petri plate with solid agar media without antibiotics
Petri plate with solid agar media with ampicillin
Petri plate with solid agar media tetracycline
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