ANTIBIOTIC RESISTANCE & BACTERIAL
TRANSFORMATION
IDENTIFICATION OF AN UNKNOWN ANTIBIOTIC-RESISTANCE GENE
INSTRUCTOR MANUAL
Robin Ulep, Rebecca Sanchez Pitre, Stephanie Messina,
Rebecca Fisher, & Jawed Alam
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ABOUT BEST SCIENCE!
Acknowledgements: The Antibiotic Resistance & Bacterial Transformation curriculum module including
laboratory kits and other support material are products of the BEST Science! program, a collaborative
project between the Academics Division of Ochsner Clinic Foundation
and the Genetics Department of Louisiana State University Health
Sciences Center – New Orleans. This program is supported by a
Science Education Partnership Award (# R25OD010515) under the
auspices of the Office of the Director of the National Institutes of
Health. The content of this manual and associated products are solely
the responsibility of the BEST Science! program personnel and do not
necessarily represent the official views of the NIH. The development
of this laboratory module was significantly aided by the knowledge
and concepts created by other individuals and those contributions are
acknowledged in the bibliography.
For more information about BEST Science! and the services and resources provided by the program, please
visit the program website www.academics.ochsner.org/bestscience or contact
Allison Sharai [email protected]
504-842-4712
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TABLE OF CONTENTS
ABOUT BEST SCIENCE! ___________________________________________________________ 2
TABLE OF CONTENTS ____________________________________________________________ 3
Antibiotics – A Brief Introduction __________________________________________________ 4
ANTIBIOTIC RESISTANCE – A BRIEF INTRODUCTION ___________________________________ 5
BACTERIAL TRANSFORMATION – A BRIEF INTRODUCTION ______________________________ 8
BIBLIOGRAPHY ________________________________________________________________ 11
IDENTIFICATION OF AN UNKNOWN ANTIBIOTIC-RESITANCE GENE COMPLETE PROTOCOL ____ 12
EXPERIMENT OVERVIEW _____________________________________________________________ 12
PRE-LAB STUDENT PREPARATION ______________________________________________________ 13
PRE-LAB TEACHER PREPARATION ______________________________________________________ 15
MATERIALS ________________________________________________________________________ 15
GENERAL PRECAUTIONS ______________________________________________________________ 16
STEP-BY-STEP PROCEDURE ____________________________________________________________ 17
Prepare for the Experiment ___________________________________________________________________ 17
Add Antibiotics to Agar Plates _________________________________________________________________ 18
Prepare Competent Bacteria __________________________________________________________________ 20
Mix Competent Bacteria with DNA _____________________________________________________________ 21
Heat Shock & Recover ________________________________________________________________________ 21
Plate Bacterial Cells __________________________________________________________________________ 22
Results & Analysis ___________________________________________________________________________ 23
DISCUSSION __________________________________________________________________ 25
APPENDIX ____________________________________________________________________ 26
A.1 TRANSFORMATION LAB – SHORT PROTOCOL __________________________________________ 26
Prepare for the Experiment ___________________________________________________________________ 26
Add Antibiotics to the Agar Plates ______________________________________________________________ 26
Prepare Competent Bacteria __________________________________________________________________ 26
Mix Competent Bacteria with DNA _____________________________________________________________ 27
Heat Shock the DNA/Cell Mixture ______________________________________________________________ 27
Plate the Bacterial Cells ______________________________________________________________________ 27
Results & Analysis ___________________________________________________________________________ 28
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ANTIBIOTICS – A BRIEF INTRODUCTION
Antibiotics are drugs that are used to treat bacterial infections in humans and animals. They can be broadly classified
into two types: bactericidal, which kill bacteria, and bacteriostatic, which stop bacteria from replicating. There are a
wide range of antibiotics available to healthcare providers, and each antibiotic has a different way in which it
interferes with bacteria to kill them or stop their growth. Figure 1 below shows some common bacterial structures or
processes that are often targeted by antibiotics.
Figure 1: Common antibiotic mechanisms of action
Within the groups of antibiotics that have a common mechanism of action, there are additional variations in the
manner in which that action is accomplished. For example, in this lab we will use Ampicillin (a Penicillin derivative),
Kanamycin (an Aminoglycoside) and Chloramphenicol antibiotics. Kanamycin and Chloramphenicol both inhibit the
construction of new proteins, termed protein synthesis. Kanamycin interacts with the ribosome, inhibiting correct
reading of the genetic code and the transfer of amino acids from the ribosomes, so that proteins cannot be
constructed. Chloramphenicol prevents the formation of the peptide bonds that are necessary to link amino acids to
make proteins. Both of these result in a stoppage in protein construction through two completely different methods.
Over time strains of bacteria have emerged that are resistant to antibiotics. Antibiotic resistance genes are the focus
of this lab and will be discussed more thoroughly in the following sections. It is important to note that a single
resistance gene only confers resistance to one antibiotic. This is due to the unique target that each antibiotic has. For
example, a Kanamycin resistant gene can make a protein that modifies the Kanamycin structure so that it can no
longer interact with the ribosome to prevent protein synthesis. This resistance protein would not be able to affect
Chloramphenicol because it would not be able to block it from interfering with peptide bond formation. So even
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though both Kanamycin and Chloramphenicol inhibit protein synthesis, the Kanamycin resistance gene allows the
bacteria to continue replicating only in the presence of Kanamycin, not Chloramphenicol.
ANTIBIOTIC RESISTANCE – A BRIEF INTRODUCTION
Development of Antibiotic Resistance: Antibiotics are the main therapeutic tools used in medicine to treat
a variety of bacterial infectious diseases from pneumonia and tuberculosis to syphilis and gonorrhea. Since
the introduction of penicillin in 1943 during World War II, an increasing number of antibiotics have been
developed with the inevitable proliferation of antibiotic-resistant bacteria. Antibiotic resistance occurs
when an antibiotic can no longer effectively control or stop bacterial growth; the bacteria become
resistant and continue to multiply despite the use of the antibiotic. Survival of the fittest naturally occurs
and selection is directed towards resistant strains of bacteria (Figure 1)1.
Mechanisms by Which Bacteria Acquire Resistance: Bacteria generally develop antibiotic resistance as a
result of genetic changes - either through mutation of their own DNA or through appropriation of
antibiotic resistance genes (ARGs) from other bacteria2. For instance, an antibiotic may kill bacterial cells
by binding to and inhibiting a protein essential for cell growth. A spontaneous mutation within the gene
encoding this protein may result in a mutant protein that still has cell growth activity but is no longer able
to bind to the antibiotic, thus rendering the cell insensitive to the antimicrobial.
ARGs typically encode either enzymes that chemically degrade or inactivate the antibiotic or proteins that
form “efflux pumps” or channels that actively export antimicrobials and other compounds out of the cell,
thus preventing accumulation of chemicals toxic to the cell. Inter-bacterial transfer of ARGs occurs in
nature typically through one of three processes: transformation, transduction, or conjugation (Figure 2).
During transformation, bacteria take up naked, “foreign” DNA from their environment (often DNA
released after death of other cells) and incorporate it into their own chromosome. Transduction is a
Figure 1. Development of Antibiotic Resistance
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process in which DNA is exchanged between two bacteria with the assistance of bacterial viruses or
bacteriophages. During viral infection, bacteriophages can acquire pieces of donor cell DNA and can then
transfer that DNA to recipient cells upon further infection. Conjugation involves the transfer of ARGs via a
bridge, called a pilus, which connects two bacteria2.
Consequences of Antibiotic
Resistance: Antibiotic resistance is a
global health issue. With increasing
numbers of bacteria resistant to
antibiotics, it is becoming significantly
more difficult and more expensive to
treat and control infections. A current
example is drug-resistant tuberculosis
(TB). In the developing world,
inadequate access to medical care and
proper treatment regimens,
availability of counterfeit drugs, and
the common practice of self-
medication have substantially
exacerbated the problem of drug
resistance. Strains of multi-drug
resistant tuberculosis (MDR-TB) and
extensively drug-resistant tuberculosis
(XDR-TB) have developed as bacteria
have become resistant to multiple
antibiotics (Figure 3)3. MDR-TB occurs
when bacteria become resistant to at
least two first-line TB antibiotics. XDR-
TB is resistance to the first-line
antibiotics and at least one of the second-line antibiotics, which makes this type of TB even more difficult
to treat. A patient who develops drug-resistant TB can then transmit the drug-resistant form to other
individuals perpetuating the cycle.
Improper use of antibiotics and the concomitant proliferation of antibiotic-resistant bacteria are not only a
concern in the developing world, but also a priority in industrialized countries. In the United States alone,
an estimated 2,049,442 illnesses and 23,000 deaths resulted from infection by antibiotic-resistant bacteria
in 20131. In many affluent nations, infections acquired in settings such as hospitals and nursing homes are
a major source of illness and death. Methicillin-resistant Staphylococcus aureus (MRSA) is the most
common cause of hospital-acquired infection. In a 2009 study, the CDC estimated that the costs of treating
healthcare-associated infections like MRSA ranged from $28.4 to $45 billion dollars annually4. Antibiotic
resistance has led not only to increased healthcare costs but also to increased treatment complications,
Figure 2. Mechanisms of Horizontal Genetic Transfer
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extended hospital stays, additional doctor visits, and a need for expensive second-line antibiotics to
replace first-line drugs that may no longer be effective.
The misuse and overuse of antimicrobials have resulted in the emergence and spread (Figure 4)1 of strains
of bacteria that no longer respond to antimicrobial therapy. Examples of misuse are prescribing antibiotics
for a viral infection, incorrect choice of medicine, incorrect dosing, and failure to finish the complete
course of antibiotics. It is essential that both industrialized and developing nations focus on preventive
measures such as improving sanitation, encouraging hand hygiene, and minimizing improper prescription
of antibiotics for use in humans, farm animals, and agriculture.
Figure 3. Percentage of New TB Cases with MDR-TB
Figure 4. Spread of Antibiotic Resistance
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BACTERIAL TRANSFORMATION – A BRIEF INTRODUCTION
Natural Transformation: As noted above, transformation is the process by which bacteria take up naked,
“foreign” DNA. This ability - coupled with replication of the foreign DNA, either independently or after
integration into the bacterial chromosome - can potentially provide a selective advantage to the
transformed bacteria if the DNA confers a desirable trait such as antibiotic resistance or the ability to
defend against viral infections. More generally, the ability to acquire exogenous DNA by transformation or
other mechanisms enhances genomic plasticity and bacterial diversity.
Artificial Transformation: Scientists have capitalized on the ability of bacteria to take up DNA to artificially
introduce defined DNA molecules with specific characteristics and at high efficiency. Artificial bacterial
transformation was a foundational technology of genetic engineering that led to the birth of the
biotechnology industry in the early 1970s. Today, scientists use bacterial transformation for multiple
research and commercial purposes. For instance, transformation is used in the biotech industry to produce
numerous therapeutic drugs such as insulin (which is used to treat type-1 diabetes) and human growth
hormone (which is used to treat children with growth failure due to insufficient endogenous growth
hormone or those with short stature as a result of Turner’s syndrome, a genetic disorder). Researchers use
transformation to make many copies of specific DNA sequences, also known as DNA cloning. This is
particularly important when specific DNAs, as may be the case with archeological specimens of extinct
organisms such as dinosaurs and Neanderthals, are available only in limiting quantities. In transformed
bacteria, DNA pieces from these sources can be preserved indefinitely and replicated as desired. The
polymerase chain reaction (PCR) now provides an alternative means for replicating specific DNA sequences
from low-abundance samples such as DNA recovered from crime scenes, but DNA cloning is still a common
and standard procedure in research laboratories. Among other applications, scientists also use
transformation when creating mutations in genes and making proteins for research applications.
Plasmids: In nature, antibiotic-resistance genes (ARGs) are frequently found on plasmids - small, circular
pieces of DNA that replicate independently of the bacterial chromosome. Through genetic engineering,
scientists have modified and optimized
plasmids for use in artificial transformation
as described above. Plasmids used in
transformation typically contain three key
genetic features (Figure 5) a Multiple
Cloning Site that consists of recognition
sequences for one or more specific
restriction enzymes and is the region where
“foreign” genetic material, such as the
human insulin gene, the human growth
hormone gene, or Neanderthal DNA, is
inserted during cloning; 2) a DNA segment
called the Origin of Replication which is
Figure 5. Structure of a Generic Plasmid
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necessary for the plasmid to replicate itself (multiply) within the bacterial cell; and 3) an Antibiotic
Resistance Gene which allows for selection of bacterial cells that have taken up the plasmid DNA during
transformation.
Transformation Procedure: The standard bacterial transformation procedure is illustrated below. It is
important that you understand the procedure before completing the lab.
For routine transformations, Escherichia coli is the most commonly used bacteria although other bacteria
can also be transformed. E. coli at the log phase of growth is used when the experiment requires maximal
transformation efficiency. Remember, bacteria normally reproduce by binary fission. The organism
duplicates its genetic material and then divides into two separate parts. During the log phase, E. coli is
dividing and bacterial number is increasing exponentially. If, however, transformation efficiency is not
critical as in the experiment described in this manual, non-log phase cells are also suitable for
transformation.
Cells are spread onto agar plates and grown in the presence of antibiotics.
Growth media is added to the cell mixture to help the cells recover from the high temperatue enviornment.
The mixture is subjected to a Heat Shock, which allows the foreign DNA to rush inside of the permeable cell membrane.
Competent bacterial cells are mixed with foreign DNA
Bacteria is combined with Calcium Choride, which we will refer to as the Transformation Reagent (Tx).
This creates competent cells, which have a permeable membrane.
Bacteria is grown and collected.
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STEP 2: Collect cells by scrapping a few colonies off of the plate
and adding to ice-cold calcium chloride.
The cell pellet is resuspended in an ice-cold solution of CaCl2 buffer. This step
makes the E. coli cells “competent” to take up plasmid DNA. How Ca++ ions
make the cells permeable to DNA is not completely understood, but they are
thought to facilitate adherence of DNA molecules to the bacterial cell surface
by one or more mechanisms. This includes 1) disruption or weakening of the
bacterial cell surface structure, 2) increasing cell membrane fluidity, or possibly
3) neutralization of the negatively-charged phospholipids of the cell
membrane and the negatively-charged DNA molecules that, under normal
conditions, would repel each other.
STEP 3: Mix cells with plasmid DNA containing antibiotic resistant gene (ARG).
STEP 1: Grow E. coli to log phase in growth media.
The plasmid DNA(s) to be transformed is mixed with the “competent” cells and the
mixture is incubated on ice for a short period of time. In general, only a small quantity
of plasmid DNA, nanogram amounts, is used in transformations.
STEP 4: Heat shock cells at 42°C.
“Heat shock” is thought to promote internalization of the DNA, possibly by creating pores in
the cell membrane and/or by creating a thermal imbalance across the membrane (higher
temperature outside the cell, lower inside). Because of the temperature differential, warm
water rushes into the cell through the pores, thereby generating the force necessary to
carry the plasmid DNA into the cell interior. After a QUICK heat shock, the cells are cooled
on ice and then brought to room temperature. Too much incubation time at 42°C can cause
cell death. If a 42°C incubator or water bath is not available or is inconvenient, heat shock
can be performed at 37°C as described in the protocol in this manual.
STEP 5: Add growth medium to cells.
Heat shock of E. coli cells at 42OC is equivalent to a high fever in humans (107.6OF).
Like humans, bacterial cells need time to recover from such an adverse condition. E.
coli cells will recover best at 37OC in the presence of nutrients (i.e., growth medium).
This is not unlike your mom giving you warm broth when you are sick. Growth of E.
coli for a period of time also allows the internalized plasmid to replicate and also
produce the antibiotic resistance protein required for survival in the next step.
Growth media acts like food for the bacteria. E.coli is a competent bacterium,
which means that it can take up extracellular DNA from its environment due to
induced membrane permeability.
STEP 6: Spread cells on antibiotic/agar plates.
After the bacterial cells have been allowed to recover, they are spread on an agar plate containing nutrients and an
appropriate antibiotic. Only cells that have taken up the plasmid DNA and synthesized the antibiotic resistance protein
will survive in the presence of the antibiotic. Each transformed cell will continue to divide and, after 20-48 hours of
growth on the agar plate, will be represented by an individual colony. Only a very small fraction of the initial bacterial
cells take up the plasmid DNA and become transformed. For most experiments, this is not an issue. Indeed, some
experiments are considered a success even if only a single colony (or transformant) is obtained on the agar plate.
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BIBLIOGRAPHY
1. Centers for Disease control and Prevention (2013, September 16) Antibiotic/Antimicrobial Resistance.
Retrieved July 30, 2015, from http://www.cdc.gov/drugresistance/about.html
2. Food and Drug Administration, Center for Veterinary Medicine (2015, April 20) Antimicrobial Resistance:
Animation Narration. Retrieved July 30, 2015, from
http://www.fda.gov/AnimalVeterinary/SafetyHealth/AntimicrobialResistance/ucm134455.htm
3. World Health Organization (2014, October 22) Global Tuberculosis Report 2014. Retrieved July 30, 2015,
from http://gamapserver.who.int/mapLibrary/Files/Maps/Global_TB_MDRcases.png
4. Centers for Disease control and Prevention (Scott II, RD, 2009) The Direct Medical Costs of Healthcare-
associated Infections in U.S. Hospitals and the Benefits of Prevention. Retrieved July 30, 2015, from
http://www.cdc.gov/HAI/pdfs/hai/Scott_CostPaper.pdf
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IDENTIFICATION OF AN UNKNOWN ANTIBIOTIC-RESITANCE GENE
COMPLETE PROTOCOL This section provides detailed instructions for carrying out bacterial transformation with plasmid DNA and
testing for acquired antibiotic resistance. Select individual steps also include helpful hints.
EXPERIMENT OVERVIEW
Patient John Doe comes in with a painful cough, high fever, chest pain when breathing, and shortness of
breath. The doctor has determined that the patient has a bacterial infection. To determine the bacteria
causing the infection, the doctor collected a sample of mucus from the back of the patient’s throat using a
sterile cotton-tipped applicator. The throat swab was sent out to the microbiology lab for culture and
analysis. In order to make the best antibiotic selection to treat the infection it is important to know the
type of bacteria causing the infection and if it is resistant to any antibiotics.
The bacterial culture did not grow very well in the lab, so plasmid DNA (bacterial DNA) was collected from
the sample instead. Here, you are provided with the unknown plasmid DNA (referred to as pDNAx) that
may or may not harbor one or more antibiotic resistant genes (ARGs). By transforming pDNAx into E. coli
and testing growth in the presence of several different antibiotics, you should be able to identify the
specific ARG(s) residing on pDNAx. With this knowledge, the patient-care team will know what antibiotic is
best used in treatment of the patient.
Clinical Observation: The patient-care team must determine to which antibiotic the infecting
bacteria is susceptible in order to efficiently treat the patient.
Scientific Question: Which antibiotic resistance gene(s) does the unknown DNA harbor?
Date: August 1, 2017 Patient Name: John Doe Patient # : 1926472
First M Last
Gender: Male Birthday: January 11, 1982 Age: 35
Symptoms: Cough
Yellowish-green mucus Fatigue Shortness of breath Slight fever and chills Chest discomfort
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PRE-LAB STUDENT PREPARATION
Bacteria are typically cultured in circular Petri Dishes in the presence of Growth Media, which acts as the
bacteria food source. Several levels of bacterial growth may be observed on your plates as shown below.
When cell growth is too dense to visualize individual colonies, the growth is often referred to as a “lawn”
of bacteria.
Draw your prediction for bacterial growth in each scenario:
SENARIO 1. Bacteria containing AMPICILLIN-RESISTANT PLASMID DNA grown in the presence of
KANAMYCIN
SENARIO 2. Bacteria containing no ARG (Antibiotic resistant gene) grown in the presence of
KANAMYCIN
SENARIO 3. Bacteria containing AMPICILLIN-RESISTANT PLASMID DNA grown in the presence of 10 X
(100 mg/ml) AMPICILIN
SENARIO 4. Bacteria containing AMPICILLIN-RESISTANT PLASMID DNA grown in the presence of 1 X (10
mg/ml) AMPICILIN
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Discussion questions:
1. Describe and determine the effect of factors in the transformation protocol that can change
transformation efficiency (the number of bacteria that take up the plasmid).
2. In this protocol, you will add antibiotic to the plates by using glass beads to spread the drug
over the surface of the plate. Knowing this, how would you interpret bacterial growth along the
plate rim in Scenario 2?
Using your discussions in the previous questions, create a simple model (flowchart, pictogram,
algorithm, etc) to help in your results analysis.
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PRE-LAB TEACHER PREPARATION
To prepare for the Transformation Lab with students, carry out the following steps 30-60 minutes before
the start of the lab:
□Step 1 Determine the number and constitution of the lab groups. This protocol is designed for 4
students per group but, depending on the number of students in the class, one group may be smaller or
larger in size.
□Step 2 If you requested the 37OC incubator with the kit, set up and plug in the incubator.
Otherwise make sure your school incubator is set to 37OC.
□Step 3 Fill the water bath (not provided) with water such that the waterline is slightly above the
top of the legs of the float rack. If the water bath is to be prepared with a beaker of water and a hot
plate, use a beaker size that will accommodate the circular, plastic float rack. If you have more than one
hot plate, set up additional baths. Turn on the water bath, and set or adjust the temperature to 37OC.
While the heat shock is most effective at 42OC, in this protocol heat shock is performed at 37OC, which is
consistent with the growth media incubation temperature. If available, you can set up a second water
bath to 42OC for the heat shock step. Do not incubate the growth media at 42 OC. □Step 4 Insert the orange Growth Medium (GM) tubes into a float rack (one tubes per group) and
place it in the 37OC incubator. If you are using a large water bath that can accommodate all the float
racks, then it is better to put the Growth Medium into a water bath. The medium will achieve and
maintain a temperature of 37OC much better in a water bath than in a dry incubator.
□Step 5 Fill plastic container(s) with ice and place them at the individual workstations. Remove the
tubes of DNA samples, antibiotic solutions, and transformation reagent from either the refrigerator or
freezer and place the appropriate number of tubes (see Table II) in the ice bucket.
□Step 6 Place the remaining supplies at each workstation and shared station as listed in Table I.
MATERIALS
All materials required for the experiment are listed in Table I.
TABLE I: EQUIPMENT, SUPPLIES AND REAGENTS
Provided in Kit
SHARED WORKSTATION
1 biohazard bag 1 tube of ethanol for used glass beads
1 plate of competent bacteria 1 box of Kim-wipe tissues
1 bag of sterile loops 1 waste container
1 bag each of small, medium & large gloves 1 microcentrifuge
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1 orange tubes of Growth Media (GM) per group (0.5 ml each)
2 float racks (for incubation of GM tubes)
1 incubator (if requested)
EACH STUDENT GROUP WORKSTATION
4 agar plates 1 microcentrifuge tube rack
1 lab marker/Sharpie 1 waste container
4 tubes of glass beads 1 clear tube of Transformation Reagent, Tx (1 ml)
1 multi fixed-volume micropipette (50-250 µl ) 1 pink tubes of placebo (PBO) antibiotic (120 µl each)
1 bag of small pipet tips 1 blue tubes of ampicillin (AMP) antibiotic (120 µl each)
2 individually wrapped 1 ml transfer pipets 1 green tube of kanamycin (KAN) antibiotic (120 µl each)
1 bucket of ice 1 brown tube of chloramphenicol (CMP) antibiotic (120 µl each)
1 float rack 1 purple tube of pDNAx DNA (5 μl; 50 ng)
Not Provided in Kit
Water bath or equivalent with thermometer
Incubator (if not requested in kit)
GENERAL PRECAUTIONS
Please make sure to take the following precautions when carrying out this experiment:
The E. coli strain provided in this kit is classified as a Risk Group 1 (RG1) Biohazard agent:
Agents that are not associated with disease in healthy adult humans. Nevertheless, it is
important to follow universal laboratory precautions and standard microbiological practice
when conducting the experiment. Please discard all bacterial waste directly into the
Biohazard bag provided and transfer all other waste into the same bag at the end of the
experimnent.
Bacteria are all around us… on our hands, in the air, etc. To avoid contamination, please DO
NOT leave agar plates exposed to the air (i.e., uncovered) any longer than necessary when
carrying out a step.
The temperature at which specific steps are conducted, or solutions and suspensions are
incubated, is very critical for the success of the experiment. Where indicated, please follow
instructions regarding temperature without deviation.
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STEP-BY-STEP PROCEDURE
This procedure contains multiple steps. It is good scientific practice to check off each step as you complete
it. Also, where appropriate, please make sure to read the “Student Tips” before starting that step.
PREPARE FOR THE EXPERIMENT
□Step 1 Make sure each of the items listed in Table I above is present at either your group’s personal
workstation or at the shared workstation.
□Step 2 Pulse centrifuge all microcentrifuge tubes with liquid.
Microcentrifuges are expensive pieces of equipment. They should only be operated when the
tubes are inserted in a balanced configuration. Aside from full-load (6 tubes), there are three
other suitable configurations as shown below. For the 3-tube configuration, all three tubes
must have the same or similar (± 10% difference) volumes. For the 2-, 4-, and 6-tube
configurations, only the tubes across from each other need to have the same or similar
volume.
Make sure the tubes are balanced when centrifuging. Balance the antibiotics against each other, since they contain similar volumes.
□Step 3 Put the tubes of Growth Media (GM) at 37°C. Place the microcentrifuge tubes with DNA,
Antibiotics and Transfection Reagent (Tx) on ice:
pink tubes (labeled PBO) - antibiotic placebo
blue tubes (labeled AMP) - antibiotic ampicillin
green tube (labeled KAN) - antibiotic kanamycin
brown tube (labeled CMP) - antibiotic chloramphenicol
purple tube (labeled pDNAx) DNA
clear tube (labeled Tx) - bacterial transformation reagent;
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It is important that the antibiotic solutions (PBO, AMP, KAN, and CMP) always stay refrigerated or on ice as they will degrade over time at room temperature or higher. It is also important that the transformation reagent be cold at the time of the experiment. The temperature of the DNA is less critical.
ADD ANTIBIOTICS TO AGAR PLATES
□Step 4 Put on gloves. □Step 5 The experimental overview is depicted in Table II. Plate A will act as a control.
TABLE II: EXPERIMENT DESIGN
Plate ID Antibiotic DNA
A Placebo (PBO) - water pDNAx
B Ampicillin (AMP) pDNAx
C Kanamycin (KAN) pDNAx
D Chloramphenicol (CMP) pDNAx
□Step 6 Now, turn the agar plates upside down so that the dish with the agar is facing up. Using a lab
marker, label the agar plates with letters A, B, C, or D, your group identifier and date. Turn the plates right-
side up and label the lid with the same letter as on the bottom dish.
The top lid of an agar plate can be easily misplaced on the wrong dish. That is why scientists always label the bottom of the agar plate.
□Step 7 Unscrew the cap from one tube containing sterile glass beads. Remove the lid from one agar
plate and gently pour out the glass beads onto the agar. Replace the cover on the agar plate. Repeat with
the remaining glass beads and remaining agar plates.
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When pouring out the glass beads, touch the open edge of the tube to the lip of the plate and tilt the tube gently to let the glass beads flow slowly onto the agar. If you decant the glass beads quickly from high above the agar, the beads will bounce off the agar. If any beads bounce off the agar onto the bench top, DO NOT put them back on the agar plate as they are no longer sterile.
In this protocol, we are using glass beads instead of a spreader because students tend to “gouge” the agar when using a spreader.
□Step 8 Retrieve the micropipette, adjust the volume to 100 µl, and attach a clean pipet tip. Open
the pink tubes and pipet up 100 µl of the PBO solution. Dispense the PBO drop by drop over different
areas of agar plate A. Place the cover back on the plate.
□Step 9 Change the tip on the micropipette. Add 100 μl of ampicillin (AMP), kanamycin (KAN), and
chloramphenicol (CMP) antibiotics to the appropriate plates (see Table II).
□Step 10 Stack the 4 plates on top of each other. Then lift the stack with both hands and shake the
plates sideways (NOT VERTICALLY!!!) for 10 seconds. Rotate the stack 90 degrees clockwise between your
hands and shake for 10 more seconds. Repeat turning and shaking until you have completed a 360 degree
turn.
The purpose of this step is to spread, with the help of the glass beads, the liquid solution over the entire agar surface. Don’t shake the
plates so hard that the beads are flying off the agar.
□Step 11 Place the plate stack, still
containing the beads, right-side up on the
bench-top.
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PREPARE COMPETENT BACTERIA
□Step 12 Carry your ice bucket to the shared station.
□Step 13 Remove a single sterile loop from the bag by grabbing the end opposite of the loop.
Gliding the edge of the loop gently on the surface of the agar, “pick up” (scrape) 10-15 colonies from the
plate (or a 1 cm patch of bacteria if individual colonies are not available).
Be careful not to scrape off any agar from the plate. If necessary, your teacher can assist you with this step. Best results are obtained if there is a visible amount of bacteria on the loop.
□Step 14 Open the clear tube containing the Transformation Reagent (Tx). Insert the loop
containing the bacteria as far into the tube as possible. Spin the loop rapidly with your index finger and
thumb until the bacterial “glob” detaches from the loop. Discard the loop into the waste container.
You may have to tap the loop against the sides of the tube to dislodge all the cells.
Note: each student group will prepare only one tube of competent cells but it will be used for all 4 plates.
□Step 15 Unwrap one of the sterile, 1 ml
transfer pipets and remove the pipet by the bulb.
Firmly attach a small pipet tip to the end of the
transfer pipet. Pipet the bacterial suspension up and
down repeatedly until the cells are completely
resuspended. Place the tube back in ice. Discard the
transfer pipet.
Hold the tube up to the light to check the cell suspension. The suspension should be cloudy but no clumps (arrows) should be visible. Best results are obtained when cells are completely resuspended and no clumps remain.
Attaching the small pipet tip with the smaller opening, in comparison to the transfer pipet alone, helps break the bacterial “glob” into smaller pieces which
21
aids in resuspension.
MIX COMPETENT BACTERIA WITH DNA
□Step 16 Attach a new tip to the micropipette and pipet up 100 µl of the cell suspension from the
clear Tx tube. Uncap the purple pDNAx tube and add the cell suspension. Close the purple tube.
□Step 17 Flick the bottom of the purple tube with a finger several times to mix the cells and DNA.
Tap the tubes to the bench-top to force all droplets to the bottom of the tube. Insert the tubes completely
into the holes of the float rack in ice.
□Step 18 Incubate the float rack/purple tube on ice for 15 minutes.
For best results, the DNA/cell mixture should be completely immersed in ice.
HEAT SHOCK & RECOVER
□Step 19 Following the 15 minute incubation on ice, immediately place the float rack into the 37OC
water-bath for exactly two minutes.
It is critical that the cells receive a sharp and distinct heat shock. Make sure the tubes are pushed all the way down in the rack so the bottom of the tubes with the cell suspension makes contact with the warm water.
□Step 20 After the “heat shock,” immediately place the float rack back on ice for an additional 2
minutes.
For best transformation results, the change from 0°C to 37°C then back to 0°C must be as rapid as possible. If available, you can use a 42°C for the heat shock (see STEP 3 in teacher pre-lab preparation for more guidance).
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□Step 21 After 2 minutes on ice, move the float rack to the bench. Remove the tubes from the float
rack, place them on a microcentrifuge rack, and uncap.
□Step 22 Collect one orange Growth Media (GM) tube from the water bath or incubator.
□Step 23 Using a transfer pipet, transfer all of the Growth Media (GM) from the orange tube into
the purple pDNAx tube.
□Step 24 Close the purple tube. While holding each tube between your thumb and index finger, mix
the solutions by turning upside down 5 times. Insert the tube into the float rack, and place the float rack
in the 37°C water bath or incubator for 20 minutes.
If available, incubation in a water bath is the better option.
PLATE BACTERIAL CELLS
□Step 25 After 20 minutes of incubation, transfer the tube to a microcentrifuge tube rack on your
bench.
□Step 26 Attach a new tip to the micropipette. Mix the purple pDNAx tube by several gentle
inversions, uncap and transfer 100 μL of the cell suspension to each of the 4 plates (A to D).
□Step 27 Stack the plates on top of each other and spread the cells by agitation as before in Step 9.
If there is enough time in the class, leave the plate stack on the bench for 5 minutes to let the liquid
absorb into the agar. If there is insufficient time, proceed to the next step immediately.
Skip the 5 minute incubation if the class is running late.
□Step 28 To discard the glass beads, invert the stack so that the glass beads fall onto the lids. One
plate at a time, pour off the glass beads into the 50 ml tube containing ethanol (EtOH). Place the lid back
on the plate and stack the plates.
□Step 29 Place the plate stack upside down (agar dish up) in the 37oC incubator.
□Step 30 Discard your gloves and accumulated waste into the biohazard bag. Wipe down the
bench-top with disinfectant, if available. Wash your hands.
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□Step 31 Write down your predictions regarding bacterial growth for each plate in Table II, under
Step 5.
RESULTS & ANALYSIS
□Step 32 Cell growth will be evident in 20-24 hours. Carefully remove the plate stack from the
incubator and transfer to your bench top.
There may be significant condensation on the lid. DO NOT TURN THE PLATE RIGHT SIDE UP just yet.
If your class does not meet the day after cell plating, don’t let the cells grow
more than 36 hours in the incubator. Remove the plates, clean the condensation as described in the next step, and store the plates at 4OC until the next class.
□Step 33 While keeping the plates upside down, carefully separate the stack into individual plates.
Working with one plate at a time, wipe off any condensation in the inside of the lid with Kim-Wipe tissues,
then invert the plates right-side up.
□Step 34 Describe your observations in Table III. Take photographs of your plates if desired.
Several types of bacterial growth you may observe on your plates are shown below. When cell growth is too dense to visualize individual colonies, the growth is often referred to as a “lawn” of bacteria.
□Step 34 After documented, dispose of all plates in the biohazard bag.
□Step 35 If requested by your teacher, answer the questions below in Table IV.
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TABLE III: EXPERIMENTAL RESULTS Directions: Describe the type of bacterial growth observed on each plate. For plates with individual colonies, count the number of colonies. It is sometimes helpful to mark the colonies on the bottom of the plate with a permanent marker as you count. If there are a large number of colonies, divide the bottom of the plate into 4 quadrants or 8 pie slices using a straight edge and a permanent marker. Count one section and multiply the number of colonies by 4 or 8 to obtain an approximate value for total number of colonies. In the circles on the left side of the table below, manually draw what you see on the top of the corresponding agar plate. On the right side, record your observations. At minimum, they should include: relative bacterial growth, count of total bacterial colonies, and color and shape of the colonies.
Plates Observations
A: PBO + pDNAx
B: AMP + pDNAx
C: KAN + pDNAx
D: CMP + pDNAx
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DISCUSSION 1. Use the model you constructed earlier to determine the antibiotic resistance of the plasmid based on your growth data.
2. Compare your results with the rest of the class. Did any groups get different results from you? What could be the cause of the differences? 3. Based on the class data, what should the patient care team do next? 4. In a mixed population of bacteria, explain how bacteria without the antibiotic resistance gene could be protected by bacteria with the antibiotic resistance gene, without transfer of the gene. 5. Explain how globalization or increased travel and trade between countries could contribute to antibiotic resistance. 6. What can you do to prevent the spread of antibiotic resistant bacteria?
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APPENDIX
A.1 TRANSFORMATION LAB – SHORT PROTOCOL
The Short Protocol contains a simplified version of the step-by-step procedure for bacterial
transformation. Scientists who have prior experience with a procedure often follow an abbreviated version
as they are doing the actual experiment. Typically, in an abbreviated procedure, the number of steps is
condensed; although here we have maintained the same number of steps as in the Complete Protocol to
avoid confusion if a student needs to go back and forth between the two protocols. Procedural hints for
both the student and teacher have been deleted.
This procedure contains multiple steps. It is good scientific practice to check off each step completed.
PREPARE FOR THE EXPERIMENT
□Step 1 Make sure all items required for the experiment are present in the class lab (see Table II).
□Step 2 Pulse centrifuge all microcentrifuge tubes with liquid.
□Step 3 Place the microcentrifuge tubes with DNA, antibiotics and transfection reagent on ice. Put
the tubes of growth medium at 37OC.
ADD ANTIBIOTICS TO THE AGAR PLATES
□Step 4 Put on gloves.
□Step 5 Label the agar plates with your group identifier and date.
□Step 6 Add sterile, glass beads to all the agar plates □Step 7 Add 100 µl of placebo (PBO) to the appropriate plates (see Table II).
□Step 8 Add 100 μl of ampicillin (AMP), kanamycin (KAN), and chloramphenicol (CMP) antibiotics
to the appropriate plates (see Table II).
□Step 9 Stack plates and spread PBO and AMP over the entire agar surface by agitation of the glass
beads.
□Step 10 Place the plate stack on the bench.
PREPARE COMPETENT BACTERIA
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□Step 11 Carry your ice bucket to the shared station.
□Step 12 Using a sterile loop, “pick up” (scrape) 10-15 colonies from the plate (or a 1 cm patch of
bacteria if individual colonies are not available).
□Step 13 Put the loop into the tube with the Transformation Reagent (Tx) and dislodge the
bacterial “glob.”
□Step 14 Completely resuspend the bacterial cells using a sterile 1 ml transfer pipet with an
attached yellow pipet tip. Place the tube back in ice.
MIX COMPETENT BACTERIA WITH DNA
□Step 15 Add 100 µl of the cell suspension to the green pEMPTY tube.
□Step 16 Add 100 µl of the cell suspension to the green pEMPTY tube.
□Step 17 Mix the cell/DNA mixture in the green and yellow tubes by tapping the bottom of the
tubes and insert in the float rack on ice.
□Step 18 Incubate the float rack/tubes on ice for 15 minutes.
HEAT SHOCK THE DNA/CELL MIXTURE
□Step 19 Transfer the float rack into the 37OC water bath and leave for exactly two minutes.
□Step 20 After the “heat shock” transfer the float rack on ice for an additional 2 minutes.
□Step 21 After 2 minutes on ice, move the float rack to the bench. Remove the tubes from the float
rack, place them on a microcentrifuge rack and uncap.
□Step 22 Collect one orange Growth Medium (GM) tubes from the water bath or incubator.
□Step 23 Using a sterile transfer pipet, transfer all of the Growth Medium from one orange tube
into the purple pDNAx tube.
□Step 24 Mix the solutions in the purple tube by gentle inversions. Insert the tubes into the float
rack and place the float rack in the 37oC water bath or incubator for 20 minutes.
PLATE THE BACTERIAL CELLS
□Step 25 After 20 minutes of incubation, transfer the tubes to a microcentrifuge tube rack on your
bench.
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□Step 26 Mix the purple pDNAx tube by several gentle inversions, uncap and, using the
micropipette, transfer 100 μL of the cell suspension to each of the 4 plates (A to D).
□Step 27 Stack the plates on top of each other and spread the cells by agitation. Leave the plate
stack on the bench for 5 minutes. □Step 28 Discard the glass beads into the 50 ml tube containing ethanol (EtOH).
□Step 29 Place the plate stack upside down in the 37oC incubator.
□Step 30 Discard gloves and other trash. Clean up bench areas.
□Step 31 Write down your predictions regarding bacterial growth for each plate in Table II.
RESULTS & ANALYSIS
□Step 32 Cell growth will be evident in 20-24 hours. Keeping the plate stack upside down, transfer
from the incubator to your bench.
□Step 33 Wipe off any condensation in the inside of the lid with Kim-Wipe tissues and invert the
plate right-side up.
□Step 34 Describe your observations in Table III. Take photographs of your plates if desired.
□Step 35 Dispose of all plates in the biohazard bag.
□Step 36 If requested by your teacher, answer the questions in Table IV.
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