ANTIBIOTIC RESISTANCE & BACTERIAL

TRANSFORMATION

STUDENT MANUAL

Robin Ulep, Rebecca Sanchez Pitre, Stephanie Messina &

Jawed Alam

2

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 asharai@ochsner.org

504-842-4712

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TABLE OF CONTENTS

ABOUT BEST SCIENCE! ___________________________________________________________ 2

TABLE OF CONTENTS ____________________________________________________________ 3

ANTIBIOTIC RESISTANCE – A BRIEF INTRODUCTION ___________________________________ 4

BACTERIAL TRANSFORMATION – A BRIEF INTRODUCTION ______________________________ 7

BIBLIOGRAPHY ________________________________________________________________ 10

USING MICROPIPETTES ____________________________________ Error! Bookmark not defined.

TRANSFORMATION LAB – COMPLETE PROTOCOL ____________________________________ 11

EXPERIMENT OVERVIEW _____________________________________________________________ 11

MATERIALS ________________________________________________________________________ 12

GENERAL PRECAUTIONS ______________________________________________________________ 12

STEP-BY-STEP PROCEDURE ____________________________________________________________ 14

Prepare for the Experiment ___________________________________________________________________ 14

Add Antibiotics to Agar Plates _________________________________________________________________ 14

Prepare Competent Bacteria __________________________________________________________________ 16

Mix Competent Bacteria with DNA _____________________________________________________________ 17

Heat Shock & Recover ________________________________________________________________________ 17

Plate Bacterial Cells __________________________________________________________________________ 18

Results & Analysis ___________________________________________________________________________ 19

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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

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

Figure 1. Development of Antibiotic Resistance

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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,

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.

Figure 2. Mechanisms of Horizontal Genetic Transfer

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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): 1) 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 in Figure 6 and

described in more detail below.

Step 1: Grow E. coli to log phase. 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. If, however, transformation efficiency is not

critical as in the experiment described in this manual, non-log phase cells are also suitable for

transformation.

Step 2: Collect cells by centrifugation. This step not only concentrates the bacterial cells but also allows

the cells to be subsequently resuspended in the solution or buffer of choice.

Figure 6. Bacterial Transformation Procedure

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Step 3: Resuspend cells in ice-cold calcium chloride. The cell pellet is resuspended in an ice-cold solution

of 50 to 100 millimolar CaCl2. 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 including 1)

disruption or weakening of the bacterial cell surface structure, 2) increasing cell membrane fluidity, or 3)

neutralization of the negatively-charged phospholipids of the cell membrane and the negatively-charged

DNA molecules which, under normal conditions, would repel each other. Divalent cations other than Ca++

have also been used for transformation and, under certain conditions, yield even higher transformation

efficiencies.

Step 4: Mix cells with plasmid DNA. 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 5: Heat shock cells at 42OC. The DNA-cell mixture is then incubated at 42OC for 1-2 minutes, a step

which significantly increases transformation efficiency. “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). Subsequent equalization of the

temperature differential is believed to occur when warm water rushes into the cell through the pores,

thereby generating the force necessary to carry the plasmid DNA into the cell interior. Bacterial cells are

incubated at 42OC for only a short time. Prolonged incubation at this temperature will lead to significant

cell death. After heat shock, the cells are quickly cooled on ice and then brought to room temperature.

For E. coli, 42OC is the optimal temperature for heat shock but successful transformation can also be

carried with heat shock at lower temperatures. If a 42OC incubator or waterbath is not available or is

inconvenient, heat shock can be performed at 37OC as described in the protocol in this manual.

Step 6: 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.

Step 7: 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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TRANSFORMATION LAB – COMPLETE PROTOCOL

This section provides detailed instructions for carrying out bacterial transformation with plasmid DNA and

testing for acquired antibiotic resistance. The complete protocol includes an overview of the experiment,

a list of materials (equipments, supplies and reagents) supplied in the kit and those required of the school,

instructions for the teacher in order to prepare for the lab, and a detailed, step-by-step procedure for the

students to follow. Select individual steps also include helpful hints for students and/or teachers. An

abbreviated version of the step-by-step procedure (the Short Protocol) is provided in the Appendix and

may be used by students who are already familiar, or have previous experience, with bacterial

transformation. Whether using either the long or short version of the protocol, for best results, please

follow instructions carefully and with fidelity. Finally, this manual also contains a table to record results

and a set of questions for the students to answer.

EXPERIMENT OVERVIEW

This experiment is designed to introduce students to the basic bacterial transformation procedure used in

research laboratories. In this experiment, students will transform an E. coli bacterial strain with either a

control plasmid that confers no antibiotic resistance (no growth in the presence of antibiotics) or with a

plasmid that contains the gene for resistance to penicillin or penicillin-like antibiotics (growth in the

presence of antibiotics). After the transformation, bacteria will be grown on agar plates either with or

without antibiotics.

The experimental overview is depicted in Table I and descriptions of the DNAs are provided in the “Step-

by-Step Procedure” section below. Before viewing the results of the experiment, students should

complete Table I by predicting the “expected results” for each plate. After completing this experiment, the

students should be sufficiently familiar with the concept and procedure of bacterial transformation to

develop a relevant scientific question and to design and carry out their own extension experiment.

TABLE I: EXPERIMENT DESIGN

Plate ID Antibiotic DNA Expected Results

A Placebo (PBO) pEMPTY

B Placebo (PBO) pBLA

C Ampicillin (AMP) pEMPTY

D Ampicillin (AMP) pBLA

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MATERIALS

All materials required for the experiment are listed in Table II.

TABLE II: EQUIPMENT, SUPPLIES AND REAGENTS

EACH STUDENT GROUP WORKSTATION SHARED WORKSTATION

Provided in Kit

4 agar plates 1 biohazard bag

1 lab marker/Sharpie 1 plate of bacteria

4 tubes of glass beads 1 bag of sterile loops

1 multi fixed-volume micropipette (50-250 µl ) 1 bag each of small, medium and large gloves

1 bag of small pipet tips 1 tube of ethanol for used glass beads

3 individually wrapped 1 ml transfer pipets 1 box of Kim-wipe tissues

1 bucket of ice 1 waste container

1 float rack 1 microcentrifuge

1 microcentrifuge tube rack 2 orange tubes of Growth Media (GM) per group (0.5 ml each)

1 waste container 2 float racks (for incubation of GM tubes)

1 clear tube of Transformation Reagent, Tx (1 ml) 1 incubator (if requested)

2 pink tubes of PBO (120 µl each)

2 blue tubes of AMP (120 µl each)

1 green tube of pEMPTY DNA (5 µl; 50 ng)

1 yellow tube of pBLA DNA (5 µl; 50 ng)

Not Provided in Kit

Waterbath 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

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Biohazard bag provided and transfer all other waste into the same bag at theend 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.

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.

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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 II above is present at either your group’s

personal workstation or at the shared workstation.

□Step 2 Place the pink, blue, green and yellow tubes in the microcentrifuge and centrifuge for 10

seconds to recover all of the liquid at the bottom of the tube.

Make sure the tubes are balanced when centrifuging. Balance the pink tubes

against each other, the blue tubes against each other, and the green tube

against the yellow tube.

□Step 3 Make sure the following items are already on ice, and if not, place them in your ice bucket:

two pink microcentrifuge tubes (labeled PBO) containing an antibiotic placebo;

two blue microcentrifuge tubes (labeled AMP) containing the antibiotic Ampicillin;

one green microcentrifuge tube (labeled pEMPTY) containing a control plasmid DNA with NO

antibiotic resistance gene;

one yellow microcentrifuge tube (labeled pBLA) containing the plasmid DNA with the Ampicillin

resistance gene, bla (beta-lactamase);

one clear microcentrifuge tube (labeled Tx) containing the bacterial transformation reagent;

one white, circular float rack with the legs completely inserted into the ice.

ADD ANTIBIOTICS TO AGAR PLATES

□Step 4 Retrieve a pair of gloves from the Shared Station and put them on.

□Step 5 Turn the agar plates upside down so that the dish with the agar is facing up. Using a lab

marker, label all the plates with your group identifier and date. Then label each plate with one of the

letters A-D as in Table I. Turn the plates right-side up and label the lid with the same letter as on the

bottom dish.

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□Step 6 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.

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.

□Step 7 Retrieve the micropipette, adjust the volume to 100 µl, and attach a clean pipet tip. Open

one of the pink tubes and pipet up 100 µl of the PBO solution. Remove the cover from plate A and

dispense the PBO drop by drop over different areas of the agar plate. Place the cover back on the plate.

Repeat with the 2nd pink tube and plate B.

□Step 8 Change the tip on the micropipette. Open one of the blue tubes and pipet up 100 µl of the

AMP solution. Remove the cover from plate C and dispense the AMP drop by drop over different areas of

the agar plate. Place the cover back on the plate. Repeat with the 2nd blue tube and plate D.

□Step 9 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 10 Place the plate stack,

still containing the beads, right-side up

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on the bench-top and leave undisturbed until required.

PREPARE COMPETENT BACTERIA

□Step 11 Carry your ice bucket to the shared station.

□Step 12 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 13 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.

□Step 14 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.

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MIX COMPETENT BACTERIA WITH DNA

□Step 15 With the tube still on ice, uncap the green pEMPTY tube. Retrieve the micropipette

(should already be set to 100 µl) and attach a fresh pipet tip. Invert the cell suspension in the clear Tx tube

several times, uncap, and transfer 100 µl of the cell suspension into the green pEMPTY tube. Close the

green tube.

□Step 16 Attach a new tip to the micropipette and pipet up another 100 µl of the cell suspension

from the clear Tx tube. Uncap the yellow pBLA tube and add the cell suspension. Close the yellow tube.

□Step 17 Remove the green and yellow tubes from the ice. Flick the bottom of the tubes 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 tubes 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, carry the ice bucket to the shared station. Grab

the float rack by the handle and immediately place it 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 two minutes of “heat shock” remove the float rack and immediately insert it

back into the ice. Incubate the tubes 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.

□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 Remove two orange Growth Medium (GM) tubes from the waterbath or incubator. Place

the tubes across from each other in the microcentrifuge and centrifuge for 5 seconds to recover all of the

liquid at the bottom of the tube.

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□Step 23 At your workstation, place the orange tubes in the microcentrifuge rack at room

temperature. Unwrap a sterile, 1 ml transfer pipet from its pouch. Pipet up all of the Growth Medium in

one of the orange tubes and transfer it into the green pEMPTY tube. Close the green tube and discard the

transfer pipet.

□Step 24 Unwrap another sterile, 1 ml transfer pipet and transfer all of the Growth Medium in the

2nd orange tube into the yellow pBLA tube. Close the yellow tube and discard the transfer pipet.

□Step 25 While holding each tube between your thumb and index finger, mix the solutions by

gentle inversions. Insert the tubes into the float rack. Place the float rack in the 37oC waterbath or

incubator for 20 minutes.

If available, incubation in a waterbath is the better option.

PLATE BACTERIAL CELLS

□Step 26 After 20 minutes of incubation, retrieve the float rack from the waterbath or the incubator

and move the tubes to a microcentrifuge tube rack on your bench.

□Step 27 Attach a fresh tip to the micropipette. Mix the green pEMPTY tube by several gentle

inversions, uncap, and aspirate 100 µL of the cell suspension. Remove the cover of Plate A and dispense

the cell suspension drop by drop over different areas of the agar. Repeat with Plate C.

□Step 28 Attach a new tip to the micropipette. Mix the yellow pBLA tube by several gentle

inversions, uncap and aspirate 100 µL of the cell suspension. Remove the cover of Plate B and dispense

the cell suspension drop by drop over different areas of the agar. Repeat with Plate D.

□Step 29 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.

□Step 30 Take the plate stack to the Shared Station. 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 31 Place the plate stack upside down (agar dish up) in the 37oC incubator.

□Step 32 Discard your gloves and accumulated waste into the biohazard bag. Wipe down the

bench-top with disinfectant, if available. Wash your hands.

□Step 33 Write down your predictions regarding bacterial growth for each plate in Table 1.

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RESULTS & ANALYSIS

□Step 34 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.

□Step 35 While keeping the plates upside down, carefully separate the stack into individual plates.

Working with one plate at a time, separate the bottom agar dish from the lid and place it upside down on

the bench. Wipe the condensation on the lid with a Kim-Wipe tissue and then put the bottom agar dish

back on the lid. The plates can now be placed right-side up.

□Step 36 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 37 Dispose of all plates in the biohazard bag.

□Step 38 If requested by your teacher, answer the questions below in Table IV.

No Bacterial Growth Full Growth Individual Colonies (Bacterial Lawn)

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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 + pEMPTY

B: PBO + pBLA

C: AMP + pEMPTY

D: AMP + pBLA

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TABLE IV: POST-LAB QUESTIONS

Question 1 a) In terms of bacterial growth, what was your expected result for Plate A?

b) Why did you expect such a result?

c) Did the actual result for Plate A agree with your expectations? If not, provide one or more

reasons why the expected and actual results didn’t agree.

Question 2 a) In terms of bacterial growth, what was your expected result for Plate B?

b) Why did you expect such a result?

c) Did the actual result for Plate B agree with your expectations? If not, provide one or more

reasons why the expected and actual results didn’t agree.

Question 3 a) In terms of bacterial growth, what was your expected result for Plate C?

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b) Why did you expect such a result?

c) Did you see any bacterial cell growth around the outside edge of Plate C? If so, what may

be the cause(s) of this cell growth?

Question 4 a) In terms of bacterial growth, what was your expected result for Plate D?

b) Why did you expect such a result?

c) How many colonies grew on Plate D? Based on this number, do you consider this

transformation experiment to be successful or unsuccessful? Explain.

d) On Plate D, did you see individual colonies surrounded by many smaller colonies? If so,

what are these smaller colonies called? What causes the growth of these smaller colonies?

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Question 5 a) Transformation efficiency is a number calculated which represents how well the bacteria

cells were transformed. It is expressed as the number of transformed bacteria per

microgram of DNA used. In this experiment, it represents the number of antibiotic resistant

colonies per microgram of pBLA used. Calculate the transformation efficiency for your

experiment.

b) Compare the transformation efficiency obtained by your group to those of other groups

in your class. Are they all the same? If the transformation efficiencies differ from one

another, what would cause this variation?