ANTIBIOTIC RESISTANCE & BACTERIAL TRANSFORMATION

ANTIBIOTIC RESISTANCE & BACTERIAL TRANSFORMATION STUDENT MANUAL Robin Ulep, Rebecca Sanchez Pitre, Stephanie Messina & Jawed Alam

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 2

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 11 TRANSFORMATION LAB COMPLETE PROTOCOL 16 EXPERIMENT OVERVIEW 16 MATERIALS 17 GENERAL PRECAUTIONS 17 STEP-BY-STEP PROCEDURE 19 Prepare for the Experiment 19 Add Antibiotics to Agar Plates 19 Prepare Competent Bacteria 21 Mix Competent Bacteria with DNA 22 Heat Shock & Recover 22 Plate Bacterial Cells 23 Results & Analysis 24 3

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. Figure 1. Development of Antibiotic Resistance 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 bacteria 2. 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 4

transfer that DNA to recipient cells upon further infection. Conjugation involves the transfer of ARGs via a bridge, called a pilus, which connects two bacteria 2. 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 selfmedication 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 Figure 2. Mechanisms of Horizontal Genetic Transfer 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 2013 1. 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 annually 4. 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. 5

Figure 3. Percentage of New TB Cases with MDR-TB 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 4. Spread of Antibiotic Resistance 6

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 Figure 5. Structure of a Generic Plasmid inserted during cloning; 2) a DNA segment called the Origin of Replication which is 7

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. Figure 6. Bacterial Transformation Procedure 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. 8

Step 3: Resuspend cells in ice-cold calcium chloride. The cell pellet is resuspended in an ice-cold solution of 50 to 100 millimolar CaCl 2. 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 42 O C. The DNA-cell mixture is then incubated at 42 O C 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 42 O C 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, 42 O C is the optimal temperature for heat shock but successful transformation can also be carried with heat shock at lower temperatures. If a 42 O C incubator or waterbath is not available or is inconvenient, heat shock can be performed at 37 O C as described in the protocol in this manual. Step 6: Add growth medium to cells. Heat shock of E. coli cells at 42 O C is equivalent to a high fever in humans (107.6 O F). Like humans, bacterial cells need time to recover from such an adverse condition. E. coli cells will recover best at 37 O C 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. 9

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 Healthcareassociated 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 10

USING MICROPIPETTES Unlike in a typical chemistry lab where volumes are frequently measured in liters and milliliters (ml) using graduated cylinders and calibrated flasks, in modern biology research laboratories, many procedures require the use of very small volumes of liquids often in the microliter (µl) range. Measurement of such small volumes of liquids, typically between 1-1000 µl (or one-millionth to one-thousandth of a liter), requires the use of specialized instruments called micropipettes. When working with these volumes, small errors in pipetting can result in large variations in the concentration of chemicals in the solution leading to inaccurate experimental data or even failed experiments. Thus for best experimental results, it is critical that students are comfortable with the use of micropipettes. Prior to conducting the experiments described in this manual, we highly recommend that students practice micropipetting small volumes of water following the instructions and tips provided below. The Lab2Go kit accompanying this manual contains a multi-fixed volume micropipette rather than the more familiar continuously variable, adjustable micropipette. It is able to measure volumes of 50, 100, 150,200 and 250 µl (Figure 7). Barrel A Volume Line Tip Ejector Volume Indicator Plunger Button & Volume Adjustor HOW TO USE THE MICROPIPETTE 1. Hold the pipette in one hand. With the other hand, turn the PLUNGER BUTTON until the VOLUME LINE points toward the desire volume (for instance, 250 µl in A). 11

2. The plunger has three stopping positions: the unpressed TOP POSITION (B), the FIRST STOP (C) and the SECOND STOP (D). All three positions are used during aspiration and dispensing of the liquid. The proper position of the plunger when carrying out these processes is essential for accurate pipetting. Please follow directions faithfully. B C D 3. Holding the pipette vertically, insert the bottom of the pipette (the TIP ATTACHMENT SITE) into a new disposable tip in the tip box (E). Press the pipette downward until it firmly attaches to the tip (F). The tip should remain attached when the pipette is pulled upwards from the tip box (G) and makes a positive, airtight seal. Alternatively, remove a tip from the box or a bag with your fingers and manually fit the tip onto the end of the pipette (H). E F G H Tip Attachment Site 12

4. Press the PLUNGER BUTTON slowly until you feel resistance. This is the FIRST STOP and corresponds to the desired volume of liquid. DO NOT PRESS THE BUSH BUTTON ANY FURTHER WHEN ASPIRATING LIQUID AS THE PIPETTED VOLUME WILL BE INACCURATE. 5. Holding the pipette vertically and the PLUNGER BUTTON pressed to the FIRST STOP, immerse the tip into the sample to a depth of 2-4 millimeters (I). Rrelease the PLUNGER BUTTON slowly and allow it to return to the TOP POSITION to draw up the liquid (J). DO NOT RELEASE THE PLUNGER BUTTON QUICKLY AS THIS MAY RESULT IN THE LIQUID BEING SUCKED UP INTO THE PIPETTE. Withdraw the tip from the sample liquid (K). I J K 6. Place the tip containing the liquid against the inside wall of the receiving vessel at an angle of 10 to 40 degrees (L). Press the PLUNGER BUTTON slowly to the FIRST STOP. This will expel most of the liquid into the vessel (M). Wait 1-3 seconds and then press the PLUNGER BUTTON completely (this is the SECOND STOP). This will expel the residual liquid from the tip (N). Withdraw the pipette from the receiving vessel and then release the PLUNGER BUTTON slowly (O). If additional aliquots of the SAME SAMPLE are to be pipetted, repeat Steps 3-6. L M N O 13

P Q 7. When pipetting is complete, position the pipette above a waste container (P) and press the TIP EJECTOR button to discard the tip (Q). Tip Ejector There are many videos describing the proper use of micropipettes. Links to some of these videos are provided below. https://www.youtube.com/watch?v=p-opoybezp0 https://www.youtube.com/watch?v=uey_ngdfo_8 https://www.youtube.com/watch?v=ngoswmrjjao Finally, the following tips are extremely useful whether using an adjustable or fixed-volume micropipette. 14

PIPETTING TIPS AND PRECAUTIONS 1. Do not use a micropipette without the proper disposable tip firmly attached to the barrel. Failure to use a pipette tip will contaminate the pipette barrel. 2. When aspirating (drawing up) a solution, push the plunger to the first stop only and lower just the pipette tip below the level of the solution that you are sampling. You should be holding the tube containing the solution in your hand about eye level. It s important to actually see the solution enter the pipette tip. 3. Slowly release the plunger and allow the liquid to move into the pipette tip. Be certain that you re not aspirating air into the tip. 4. When dispensing (pushing out) the liquid, place the pipette tip into the tube that will receive the solution. Position the tip so that it touches the side and near the bottom of the tube. Slowly push down on the plunger to the first stop and then push all the way to the second stop until all of the solution is released. Keep your thumb pressed on the plunger and remove the pipette from the tube into which you re dispensing the liquid. This will avoid reaspirating the liquid into the pipette tip. Be certain that you see the solution leaving the tip. Release the plunger once the pipette is outside of the tube. 5. When dispensing a new reagent, always use a fresh tip to avoid contamination. 6. Do not lay down a micropipette with fluid in the tip or hold it with the tip pointed upward. Fluid could leak back into the pipette. 7. Avoid letting the plunger snap back when withdrawing or ejecting fluid; it will eventually destroy the piston. 15

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 Stepby-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 16

MATERIALS All materials required for the experiment are listed in Table II. TABLE II: EQUIPMENT, SUPPLIES AND REAGENTS EACH STUDENT GROUP WORKSTATION Provided in Kit SHARED WORKSTATION 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 17

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. 18

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. 19

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 2 nd 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 2 nd 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 20

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. 21

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 37 O C 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. 22

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 2 nd 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 37 o C 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 37 o C 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. 23

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. No Bacterial Growth Full Growth Individual Colonies (Bacterial Lawn) Step 37 Dispose of all plates in the biohazard bag. Step 38 If requested by your teacher, answer the questions below in Table IV. 24

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 25

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? 26

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? 27

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? 28