Introduction to Genetic Engineering. Bacterial Transformation with Green Fluorescent Protein (pglo) Teacher Guide

Transcription

1 Introduction to Genetic Engineering Bacterial Transformation with Green Fluorescent Protein (pglo) Teacher Guide Table of Contents Teacher Guide Letter to Teacher..I Inventory Sheet...II Suggested Transformation Lab Preparation Sequence.....IV Laboratory Preparation Instructions V Important Laboratory Concepts Covered......VIII Appendix to Teacher Guide.. X Bacterial Transformation Lab Activity Introduction Background Information and Scientific Theory. 2 General Lab Skills Required 4 Laboratory Activity Worksheet: Bacterial Transformation Worksheet: Calculating Transformation Efficiency Acknowledgements..15

2 Dear Teacher, One of the biggest challenges for first-time students of biotechnology or molecular biology is that many of the events and processes they are studying can t be seen by the human eye. A great way to provide an enlightening learning experience for these students is to use the gene from a bioluminescent jellyfish, Aequorea victoria, that makes green fluorescent protein (GFP). GFP fluoresces a brilliant, glowing green when viewed under UV light and its presence is easily observable. In this lab, your students will perform a procedure known as genetic transformation. Genetic transformation occurs when a cell is given a new piece of genetic material (DNA). This new genetic information often provides the organism with a new trait, which is identifiable after the transformation process. When a cell is genetically transformed, functional alterations may be caused by the direct uptake of new genes into an organism s genome. These traits can be observable, as in the case of GFP. Genetic transformation is also called Genetic Engineering, or Recombinant DNA Technology - the revolutionary discovery that launched the biotechnology industry. The technique is especially useful because specific genes can be excised from human, animal, or plant DNA and inserted into bacteria or other cells, to give them new functions. For example, the DNA sequence for the human hormone insulin can be put into bacteria. Under the right conditions, these bacteria can then make human insulin and patients with diabetes can be treated with this recombinant insulin. This process has increased the quality and accessibility of treatment for diabetes patients worldwide. Genetic transformation is used in many areas of biotechnology. In agriculture, genes coding for traits such as frost, pest, or drought resistance can be genetically transformed into plants. In bioremediation, bacteria can be genetically transformed with genes enabling them to digest the oil from oil spills. In medicine, gene therapy on humans, or genetically transforming a person with a disease caused by defective genes has not been as successful. Studies to reverse blindness and bleeding disorders have been promising but still require more research. Reading through this Teacher Guide carefully will guide your success in the classroom. Included are the following: Inventory Sheet. Please check to see that all the materials listed on the Inventory Sheet are available from BABEC and store each item appropriately. Make sure that you have all the needed items not provided in the BABEC kit. Suggested Transformation Sequence: Use this information to plan the layout of the lab in your class. Laboratory Preparation Instructions: Read these instructions carefully for suggestions on setting up this activity in your classroom. Important concepts and background information Teacher s Version: The Teacher s Version of the lab manual includes both the Student Version and important notes at points in the protocol where mistakes may occur. Please make sure to read the lab manual carefully and closely monitor students techniques during these steps. It includes an answer key. Questions? Please contact us at babec@babec.org. Please Note: We re interested in hearing your thoughts on this curriculum. We will be making changes to the curriculum to better integrate NGSS. Please let us know how we might improve this curriculum we want to hear your ideas! I Bacterial Tansformation: Teacher Version General April 2016

3 Inventory Sheet Listed on the following table are the reagents and consumable supplies provided in the pglo Bacterial Transformation Kit from BABEC. If you need access to additional materials, please contact us. For this lab, a team is considered to be a group of 2 students. Each group will prepare 3 bacteria plates (with plasmid on LB, LB/amp, LB/amp/ara) and 2 teams will also include negative control plates (no plasmid on LB and LB/amp). Depending on your room configuration and number of students, it might make sense to divide the students into groups of 3-4 and assign two students for the DNA negative control plates, while the other two perform the +DNA procedure. The Transformation Kit supports 16 lab stations (a station of 2 students or a total of 32 students) and some overage for aliquoting. pglo Transformation Reagents provided in the BABEC Kit: Item Storage Amount Per Kit Amount Per Team Live E. coli culture plate Refrigerator (4 C) 1 plate 1 colony 10 ng/µl pglo plasmid, 250uL/tube Freezer (-20 C) 1 tube 10µL Transformation solution (TS) 50mM CaCl2 Room Temp (20 25ºC) 20mL 1mL LB Agar Refrigerator (4 C) 3 bottles at 250 ml/ bottle Ampicillin (10 mg/ml) Freezer (-20 C) 3 ml Arabinose (200 mg/ml) Freezer (-20 C) 3 ml Enough to make 20 plates of each LB, LB/amp, LB/amp/ara LB nutrient broth, sterilized Room Temp (20 25ºC) 20mL 1mL NOTE: If you do not use the frozen reagents by the end of the school year, please contact BABEC to determine if they should be discarded. You should have some left over after each reagent is aliquoted. Please keep this as stock for emergency needs in the class. II Bacterial Tansformation: Teacher Version General April 2016

4 pglo Transformation Equipment & Supplies needed at the school site: Item Comments Microwave oven & oven mitts Sterile Petri dishes (60mm small) For melting the LB/Agar for preparing plates 3 per group plus 4-8 extra plates for negative controls; plates Black, red, green thick markers To mark plates edges when pouring plates Crushed ice Styrofoam cups Water bath Thermometer that can read 42 o C/55 o C Distilled water Foam microtube holder/float Timer Incubator oven 1.5mL microtubes Microtube racks UV light source Safety goggles Waste containers or beakers Permanent markers Disinfectant Micropipettor (P20) Inoculation loops (10µL) - optional Micropipettor (P1000) Micropipette tips (P20) Sterile Disposable gloves For chilling E. coli cells before heat shock. For holding crushed ice at work stations. Set to 42ºC for heat shock step. Set to 55 o C when keeping LB agar for plate pouring For filling water bath; prevents contamination. To keep tubes floating in the water bath. To keep track of the heat shock time. To grow E. coli cells at 37ºC overnight. (alternatively, grow on benchtop at room temperature for 2-3 days) 1-2 per team (optional: use 4 different colored tubes to prevent mix-ups) 1 per team Transilluminator or hand-held lamp To protect eyes when looking at UV light For disposal of pipettes, loops, tubes, etc. 1 per team 10% bleach or Amphyl; to disinfect any surface that contacted the bacteria 1-2 per team (alternatively you can use disposable sterile plastic transfer pipettes) 2-4 per team in place of micropippetors to deliver 10µL and for spreading To deliver ampicillin and arabinose when pouring plates 1 box per station Optional III Bacterial Tansformation: Teacher Version General April 2016

5 Suggested Transformation Lab Preparation Sequence Activity When Prep Time * 1 Read manual, create a lesson plan. Make copies of Student Guide and worksheets. Before starting lab prep 2 hrs or more 2 Do only if you are not provided with these in advance (refer to page V-VI for instructions): Prepare LB Prepare LB agar plates. Prepare and sterilize TS, LB agar and LB broth. 3-7 days prior to student use 1.5 hrs or more 3 Streak student team starter plates using an inoculation loop and the E. coli culture plate. Alternatively, have students create their own team starter plates from the E. coli culture plate. (Refer to page VII for instructions) Aliquot 1mL of LB broth and 1mL of TS for each team into sterile, labelled microfuge tubes (color coded if possible) hrs prior to student use 30min or more 4 Set-up student lab stations. Fill hot water bath with distilled water and set to 42 C. Obtain crushed ice and store in nearby freezer. Night before or prior to start of lab 1 hr * Time needed depends on number of stations and class sections. Outline for Class Implementation Day Activity 1 Introduction to transformation 2 Lab - Transformation and streaking plates 3 Transformation data collection and analysis 4 Extension activity: Determining transformation efficiency 5 Extension activity: Student group presentations, Bioethics To gain the most from this activity, students should already be familiar with the relationship between genes and proteins, basic bacterial parts and their function, aseptic technique and micropipette or Pasteur pipet use. These concepts and techniques can also be approached during this activity, but will lengthen the time you devote to it. IV Bacterial Tansformation: Teacher Version General April 2016

6 Preparation of agar plates Laboratory Preparation Instructions Prepare LB agar plates 3-7 days prior to doing lab with your students. These plates should be left out (cured) at room temperature for two days and then refrigerated until use. Curing evaporates off the excess moisture from pouring the plates. Refrigerate as a stack, with the bottom up, in their original plastic sleeves. Tape the sleeves closed to prevent accidental opening. It is best to refrigerate vertically rather than horizontally to prevent the introduction to microbes to the plates. Item- Provided by BABEC Additional items needed ml LB Agar bottles (provided by BABEC) Microwave oven 1 tube Ampicillin (10 mg/ml, 3mL total) Permanent markers (red, green and black) 1 tube Arabinose (200 mg/ml, 3mL total) Serological pipettes 80 small Petri dishes (60 x15 mm) Heat the LB/Agar: Work with one bottle at a time. Loosen the cap of one of the LB Agar bottle. Heat the bottle in the microwave for 3-5 minutes. Watch the bottle carefully, as soon as you see the agar boiling, stop the microwave and swirl the bottle gently before putting it back in the oven. Use caution and oven mitts as you continue to heat and swirl the mixture in the microwave until it is completely liquefied (make sure there are no lumps of agar). LB broth and agar will turn from cloudy to clear (see through) and have a golden tint. The more times you cook the LB, the darker amber the coloring becomes. It is still good to use. Cool each bottle to approximately 50 o C on bench top or in a 50 o C water bath, (comfortable to hold by touch but it has not solidified do not use a thermometer to check.) Color Code Striping Plates: As you wait for the bottles to cool so they are comfortable to hold (approximately 55 C but the agar is still liquefied), label your plates (20-25) with permanent markers as specified below. Label with their contents or a striping code on the side of the plates. See sample striping on plate below. Be sure both the top and bottom halves of each plate have the proper striping. Number of plates and their contents needed for one class: Agar Mixture LB LB/amp LB/amp/ara # of Plates *^20 *20 *20 Symbol 1 line Black 2 lines Black & red 3 lines Black, red & green *Note that 20 plates will give you extra plates per type. ^You will also need extra LB plates for making initial team starter plates or to keep extra starter plates for yourself. Pouring agar plates (LB, LB/amp, & LB/amp/ara) Pull out the vials of ampicillin and arabinose from your freezer and refrigerator and place the vials into a Styrofoam cup with ice. If the vial of ampicillin hasn t defrosted by the time you need it, roll the vial between your palms to create friction to warm the vial (quick thaw). Thaw, invert to mix, and chill tubes on ice. DO NOT add ampicillin or arabinose to hot agar - both substances are unstable at high temperatures. Pouring the LB Agar only plates: Note that a quick hand in pouring is more important than flaming to reduce the introduction of microbes. Stack and spread out LB plates along the edge of a sterile counter. Stack them 3-4 high depending on how many your one hand can comfortably fit over and manipulate the stack. Swirl the bottle first. Tilt the bottom plate lid open just enough for you to pour the contents from the bottle. Using one of the LB Agar bottles, fill each plate about 1/3 full of liquid agar and replace the plate lid. You ll notice as you pour that it will flow to cover the plate. Only a thin layer of LB agar is needed to cover the bottom of the dish. If you notice air bubbles or V Bacterial Tansformation: Teacher Version General April 2016

7 that the liquid agar hasn t evenly spread throughout the plate, gently swirl, rotate the dish, avoiding splashing agar on the lid of the dish. Your agar may need reheating in a microwave or on a hot plate to liquefy the LB agar again, if it cools below 37 C. Pouring the LB Agar/Amp plates: To the cooled to touch 2 nd 250mL LB Agar, add 1.25 ml of ampicillin to the LB agar in the bottle and swirl to mix the contents. Ready the stripe-labeled plates of LB/amp in mini-stacks along the edge of a counter and pour this mixture of LB/amp until each of these plates is approximately 1/3 full. The final concentration of Ampicillin in each plate is 50µg/mL. Pouring the LB Agar/Amp/Ara plates. Now, ready LB/amp/ara stripe-labeled plates along a counter. To the cooled to touch, 3 rd bottle of LB agar add 1.25 ml of ampicillin and 2.5 ml of arabinose to the bottle. Swirl to mix and pour the flask s contents into these last plates. If you still have agar remaining in the flask, you might want to fill your own empty plates for use as occasional student errors, or for future experiments that may arise. (Please note that ampicillin and arabinose decompose over time). The final concentration of ampicillin is 50µg/mL and arabinose is 2mg/ml. Please note that once ampicillin and arabinose have been added, DO NOT reheat the agar! Ampicillin and arabinose will break down at high temperatures! Plates can be restacked after 30 minutes or when solidified. After the plates have cured for 1-2 days on a lab counter, invert and place them into their plastic sleeves for refrigeration until their use. Make sure to refrigerate vertically, bottom up rather than horizontally. Aliquoting LB Broth and Transformation Solution (TS) Label: mL microfuge tubes with LB broth mL tubes with TS and place them into a microfuge tube rack. (You might want to use different colored microfuge tubes to further differentiate these solutions.) Using a P-1000 micropipettor or a serological pipette aliquot 1mL of LB broth per team into the LB broth tubes. Transfer 1mL of TS into the 16 tubes labeled TS. Each student team should end up with 2 microfuge tubes, each with 1mL of the two solutions. Refrigerate aliquots until needed. Streaking starter plates of E. coli Starter plates are needed to produce bacterial colonies of E. coli on agar plates. LB agar plates should be streaked to produce single colonies and incubated at 37 C for hours before the transformation investigation begins. Under favorable conditions, one cell multiplies to become millions of genetically identical cells in just 24 hours. There will be millions of individual bacteria in a single millimeter of a bacterial colony. Depending on time, you may prefer your students to learn how to streak their own plates for individual colonies. Plate Streaking Streaking takes place sequentially in four sections. The first streak spreads out the cells. In subsequent streaks the cells become more and more dilute, thus increasing the likelihood of producing single colonies. VI Bacterial Tansformation: Teacher Version General April 2016

8 1. Draw quadrants on the underside of the petri dish. Using a sterile inoculation loop or sterile pipet tip, pick up one bacterial colony from live E. coli culture plate. 2. Using a back and forth motion, gently spread the colony into one quadrant of the LB starter plate. Keep the lid slightly tilted open - only as much as necessary. Be careful not to puncture the agar. 3. Rotate the plate one-quarter of a turn. Go into the previous streak about two times and then back and forth as shown for a total of about 5-10 times. 4. Again, rotate the plate one-quarter of a turn and pass over a previous streak from the previous quadrant several times with the loop. 5. Repeat step 3, but this time, drag out the loop to form a tail not touching any previous streaks. Close your plate to avoid further contamination. 6. Place the used loop (or tip) in a disinfectant solution waste cup. Follow this procedure for the remaining starter plates. Once starter plates are inoculated, incubate them upside down in a 37 C incubator oven for 24 to 36 hrs. 7. If your students are not using the plates right away, seal the sides with Parafilm or lab tape so they don t dry out, invert the plates, and place them in a dark cupboard until needed. Avoid refrigerating your starter plates as cooling will reduce your transformation efficiency. What to expect the next day You should see individual bacterial colonies in quadrant 4, and very dense bacterial growth in quadrant 1. Quadrants 2 and 3 will have bacterial density somewhere in between, similar to what is seen below: Your streaked plate should look similar to this image after hours: Note: the images on this page have been provided by the Florida Institute of Technology VII Bacterial Tansformation: Teacher Version General April 2016

9 Important Laboratory Concepts Covered Lab Safety and Disposal If you do not have an autoclave readily available, all solutions and equipment coming into contact with the bacteria such as the pipet tips and inoculating loops, should be collected and placed into disinfectant solution, such as 10% bleach. Cover each contaminated petri dish with disinfectant solution and let sit for at least 15min before disposal according to your site guidelines. For your protection, wear safety glasses and a lab coat when handling concentrated and dilute disinfectant. Make sure there is adequate ventilation. If your school has specific safety and disposal policies please follow their procedure. The E.coli used in this lab E.coli is a species of bacteria found everywhere in our environment. The strain we use for this lab and in many research labs is harmless to humans and is NOT pathogenic. They have been specially engineered to help scientist with their work. If you touch the bacteria with your hands, simply wash with soap and water. If you get some bacteria in your eyes, simply flush with water. As always, use safety precautions when working in the laboratory. Media and Additives LB (Luria-Bertani) agar and broth contain a yeast extract with a mixture of amino acids, carbohydrates, salts and vitamins. Together, these substances support bacterial growth. Agar contains a gel derived from seaweed that solidifies at room temperature. If you have extra prepared dishes, allow your students to touch the surface of one of the LB plates and help them make connections between agar and Jello. Including the antibiotic ampicillin in the media prevents the growth of bacteria other than the successful transformants. The pglo plasmid contains a beta-lactamase gene, which allows the transformed bacteria to produce an ampicillin inactivating protein, the enzyme beta-lactamase. Using ampicillin in the media insures that only bacterium containing and producing beta-lactamase will grow. Seeking survival of only the transformed cells is an example of antibiotic selection. Ampicillin breaks down over time and is sensitive to heat and repeated freeze/thaw cycles. The addition of the carbohydrate sugar arabinose in the media will activate the transcription of the GFP gene. Translation can then follow, resulting in expression of the GFP protein. The GFP protein allows the transformed cells to appear neon green when illuminated with a long-wave UV lamp or standard UV transilluminator. Without arabinose in the media, the GFP gene will not be transcribed, and the GFP protein will not be produced. The phenotypic expression of the wild-type bacteria is white. Transformed cells will also appear white when the growth media lacks arabinose, but they will fluoresce green under UV light, if arabinose is present. This engineered pglo plasmid allows students and teachers to easily verify their transformation success. Try this: If you have extra arabinose (and the time), add at least 100µl of the arabinose in a drop-wise fashion onto the bacteria growing on a previously inoculated LB/amp +DNA plate. Cover, wait a couple of minutes to allow the arabinose liquid to soak into the agar, invert, and incubate this plate for hrs to demonstrate how the GFP gene can be switched-on by the presence of arabinose in its environment. Transformation Solution (TS), 50mM CaCl 2 When fully intact, the bacterial cell membrane does not allow DNA to pass through it, so how do we get the DNA inside during transformation? We add Ca 2+ cations, which neutralize the negative charges of both the DNA phosphate-backbone and the phospholipids within the cell membrane. By neutralizing these repulsive negative charges, the DNA can then (easily) pass across the bacterial cell membrane. It is possible to get transformants if CaCl2 is missing. However, the efficiency (number of colonies on plates) might be very low. A great animation of the process is available at > Manipulation > Techniques > Transferring & Storing > Transformation Animation. VIII Bacterial Tansformation: Teacher Version General April 2016

10 Heat Shock Heat shock helps bacterial cells take in small foreign DNA segments such as plasmids by increasing a cell membrane s permeability. Students must carefully follow the pre-optimized process laid out in this protocol; it contains specific temperatures and incubation times that will ensure success. Otherwise, few, if any, bacteria will uptake the plasmid and be transformed. A great animation of the process is available at > Manipulation > Techniques > Transferring & Storing > Transformation Animation. Recovery The 10min incubation period in nutrient LB broth after the stress of heat shock allows the transformed bacteria cells to heal and grow. They will also begin to secrete beta-lactamase, the ampicillin inactivation enzyme, which increases the survival rates of the transformed cells on the ampicillin plates. Incubation Optimal growth for E.coli occurs at 37 C. E. coli will require more time to grow and express the GFP gene if kept at room temperature. A warm spot on top of the refrigerator or heating units in the classroom will help. It will take 2-3 times longer for the bacteria to grow at room temperature versus an incubator set at 37 C, but they will grow in these conditions. After the colonies appear, keep the plates by wrapping them in parafilm and placing them in the refrigerator. Satellite colonies will start to grow on the plates if the plates are left in the incubator for >36 hours. This may lead to more questions by students, creating another opportunity for a phenomena type observation. Antibiotic Selection The pglo plasmid, which contains the GFP gene, also contains the gene for beta-lactamase. Beta-lactimase is an enzyme that provides resistance to the antibiotic ampicillin, a member of the penicillin family. The beta-lactamase protein is produced and secreted by bacteria that contain the plasmid. Beta-lactamase inactivates the ampicillin present in the LB nutrient agar to allow bacterial growth. Only transformed bacteria that contain the plasmid and express beta-lactamase can grow on plates that contain ampicillin. Only a very small percentage of the cells successfully take up the plasmid DNA during heat shock and are transformed. Untransformed cells cannot grow on the ampicillin selection plates. In order to "stably retain" the plasmid, there needs to be some type of metabolic reason for the E. coli to keep the plasmid around. If the plasmid contains a gene that codes for a protein that protects against antibiotics, then only cells that have the plasmid will survive in the presence of that antibiotic Aseptic technique When growing bacteria in culture, it is important to prevent the growth of unwanted microorganisms in the nutrient rich media. Aseptic technique is a series of methods that are used to minimize the chances of contamination. Examples include use of sterile tubes and pipettes, sterilized solutions, cleaning the work area with disinfectants, use of Bunsen burners, and keeping the caps of tubes, plates and pipette boxes closed. Using student workstations It is recommended that students work in groups of two. It is up to the discretion of the teacher as to whether students should access common buffer solutions and equipment or whether the teacher should aliquot solutions in the microtubes provided. We recommend assigning 2 student groups in each class to perform the negative transformation, in addition to the pglo transformation. IX Bacterial Tansformation: Teacher Version General April 2016

11 Appendix to Teacher Guide If you are not provided with the BABEC solutions and wish to make your own use the following protocols. Preparation of agar plates and LB broth Prepare LB agar plates 3-7 days prior to doing lab with your students. These plates should be left out (cured) at room temperature for two days and then refrigerated until use. Curing evaporates off the excess moisture from pouring the plates. Refrigerate as a stack in their original plastic sleeves. Tape sleeves closed to prevent accidental opening. Materials Required for preparing LB broth and LB agar plates: Item Item LB Agar Oven mitt LB Broth Autoclave or microwave Distilled water Permanent markers (black, red and green) ml bottles (safe for autoclave/microwave) Serological pipettes 10 mg/ml Ampicillin (3ml, store 20 C) of 60x15mm Petri dishes 200 mg/ml L-(+)-Arabinose (3ml, store at 4 C) 0.2µm filters for sterilizing LB liquid medium: Lysogeny broth also known as Luria-Bertani medium is commonly used to grow bacteria. General recipe for preparation of 1 liter LB is 10g bacto-tryptone, 5g yeast extract and 5g NaCl which maybe slightly different in different recipes. You may purchase already mixed components in form of powder. Into a 100mL glass bottle, following manufacturer s instructions, measure enough LB powder and mix in 100mL of water. Autoclave or use a microwave. LB contains concentrated amount of protein and nutrients, such liquids will become super-heated when boiling. Wear an oven mitt that is long enough to cover your wrist and arm. Avoid splatters of the super-heated LB liquid that would burn your arm! Ampicillin (200X): Dissolve 30 mg of Ampicillin in 3mls distilled water. You may sterilize by filtering through a 0.2µm filter. Store in the freezer. The stock concentration is 10mg/mL. The final concentration used per plate is 50µg/mL. Arabinose (100X): Dissolve 400 mg Arabinose in 2 mls distilled water. Arabinose may need at least 10 minutes to dissolve. Filter sterilize through a 0.2µm filter. Store in the refrigerator. The final stock concentration is 200mg/mL. The final concentration used per plate is 2mg/mL. LB agar for plates: LB agar can be purchased as powder or tablets. Ready three 500mL bottles. Add 250mL distilled water in each bottle/ flask. Following manufacturer s instructions, measure enough LB agar for 250mL each. Mix and autoclave or microwave (see microwave alternative). Cool each bottle to approximately 55 o C, (comfortable to hold by touch but it has not solidified do not use a thermometer to check). Microwave alternative: Ready three 500mL bottles. Add 250mL distilled water to each bottle/flask. Following manufacturer s instructions, measure enough LB agar for 250mL each. To avoid water loss due to evaporation, place a small Erlenmeyer flask upside down into the mouth of the larger one and heat the liquid until boiling. Use caution and oven mitts as you continue to heat and swirl the mixture in the microwave until it appears that you ve boiled it to death, but avoid burning your hands! LB broth and agar will turn from cloudy to clear (see through) and have a golden tint. The more times you cook the LB, the darker amber the coloring becomes. It is still good to use. Cool each bottle to approximately 55 o C, (comfortable to hold by touch but it has not solidified do not use a thermometer to check.) X Bacterial Tansformation: Teacher Version General April 2016

12 Color Code Striping Plates: Please see page V. Pouring agar plates (LB, LB/amp, & LB/amp/ara) Please see page V-VI. DO NOT add ampicillin or arabinose to hot agar - both substances are unstable at high temperatures. Transformation Solution (TS): 50mM Transformation solution is CaCl2 (111 g/mol) dissolved in distilled water and filter sterilized. To make 100 ml TS, in a 250 ml culture bottle, weigh out 0.55 g CaCl2 and fill with 100mL distilled water. Sterilize using a filter. This should not be autoclaved or microwaved. Store aliquots at room temperature. Alternatives to micropipettes Using Transfer Pipettes If micropipettes are not available, disposable sterile plastic pipettes maybe used instead. A transfer pipette works just like an eye-dropper. Observe the 100µl, 200µl and 300µl marks on the transfer pipette. You will need to transfer a volume of 250µl, found between 200µl and 300µl. You will also need to transfer volume of 150µl between the 100µl and 200µl marks.. When using, bring the pipette up to eye level to confirm that liquid has been transferred correctly. For practice, get a feel for the pipette by transferring water from one container to another. Success of the lab depends on the proper use of tools and reagents required for the protocol. Using Inoculating loops You can measure precisely 10µl with a 10µL inoculation loop. Dip the loop into the tube containing liquid. A noticeable film will form around the ring due to surface tension (like a bubble wand). Swirl the loop into the tube to be transferred to. Note that the loop does not fit into the narrow bottom end of most microtubes. Use a 2 ml tube with a wider bottom. XI Bacterial Tansformation: Teacher Version General April 2016

13 Introduction to Genetic Engineering Bacterial Transformation with Green Fluorescent Protein (pglo) Genetic engineering is an umbrella term that encompasses many different techniques for moving DNA between different organisms. Transformation is the process by which an organism acquires and expresses a whole new gene. In this activity, you will have the opportunity to genetically transform bacteria cells; altering them so that they can make an entirely new protein. This procedure is used widely in biotechnology laboratories all over the world, enabling scientists to manipulate and study genes and proteins in exciting new ways. Adding a new gene to bacteria cells has become a relatively simple process. You will add a gene that codes for Green Fluorescent Protein (GFP). This protein was discovered in the bioluminescent jellyfish called Aequorea victoria, a jellyfish that fluoresces and glows in the dark (Figure 1). The gene for GFP was isolated in 1994 and was quickly used in laboratories as a way to brightly label proteins in a living cell. This tagging of proteins allowed researchers to visualize the location of specific proteins to learn more about their biological functions in exciting new ways. The discovery of GFP proved to be so important that the Nobel Prize in Chemistry in was awarded to Osamu Shimomura, Marty Chalfie and Roger Tsien in 2008 for their work. Since then, Roger Tsien s laboratory at UCSD has altered the GFP gene to make a full rainbow of proteins. Figure 2 shows how bacterial expressing many different colored fluorescent proteins can be grown together on one plate. Figure 1 Figure 2 Aequorea victoria A rainbow of fluorescent glowing under UV light growing on an agar plate Bacteria are commonly used for genetic transformation experiments because they are simple, single-celled organisms that grow and reproduce very quickly. Bacteria cells store their DNA on one large, circular chromosome. But they may also contain one or more small circular pieces of DNA called plasmids. Plasmids are able to replicate independently of the large bacterial chromosome, and can transfer easily between cells. Figure 3 shows the circular DNA chromosome and plasmid DNA inside of a cell. Figure 3 Genetic material in bacteria takes 2 forms Bacterial evolution and adaptation in the wild often occur via plasmid transfers from one bacterium to another. An example of bacterial adaptation is resistance to antibiotics via the transmission of plasmids. This natural process can be modified by humans to increase our quality of life. In agriculture, genes are added to help plants survive difficult climatic conditions or damage from insects, and to increase their absorption of nutrients. Toxic chemical spills can often be bioremediated (cleaned-up) by transformed bacteria specifically engineered to do the task. Currently, many people with diabetes rely on insulin made from bacteria transformed with the human insulin gene. Scientists use transformation as a tool to study and manipulate genes all the time. 1

14 Background Information and Scientific Theory The Central Dogma of Molecular Biology A basic tenet of biology, from single-celled bacteria to eukaryotes, is the mechanism of coding, reading and expressing genes. The central dogma of molecular biology states that: DNA > RNA > PROTEIN > TRAIT. This curriculum is an example of the central dogma in action. The instructions for GFP production are encoded in the DNA. When transcription is turned on, the cell turns those instructions into an mrna transcript. This transcript is then translated into protein, which provides the trait of fluorescence. Gene Regulation Every cell in the human body shares an identical genome that contains over 20,000 different genes. But if all cells have the same genes, how is it that a muscle cell ends up being so very different from a brain cell? The answer lies in the fact that there is a specific process for controlling which genes are turned on and which are turned off in every single cell. Gene regulation is the name for all the different cellular processes that have to take place in order for a gene to result in a protein. Gene regulation is an important concept in biology dictating where and when genes are turned on or off. Gene expression occurs when genes are turned on, resulting in the expression of proteins the workhorses of the cell. Proteins called transcription factors are frequently used by cells to turn transcription on or off depending on environmental conditions. They are important for cellular development, tissue specialization, and organismal adaptation to the environment. Transcription factors act at the promoter region in front of a gene. At the promoter, RNA polymerase initiates transcription and turns a gene on; the gene is then said to be expressed. Once the mrna transcript is made, it can be translated into protein (see Fig 5). All the genes in our bodies are highly regulated to allow for maximum efficiency, and to decrease waste (energy) in our cells. The pglo System In this laboratory activity, you will have the opportunity to genetically engineer a cell and you will see with your own eyes the critical role of gene regulation in living systems. This is because the expression of the GFP gene in this experiment is not automatic. Rather, it happens only when the environmental conditions are just right. Plasmids used by molecular biologists are named with an acronym that begins with the lower case "p", and followed by a name that conveys information about its function. pglo is the name for a plasmid that has been engineered to contain the gene for GFP, which glows under UV light. Using recombinant DNA technology, scientists designed this plasmid to contain two additional genes, for a total of three genes whose function it is important to understand before beginning this activity. Figure 4: The pglo plasmid has been engineered to express 3 genes amp r ara ara Codes for the regulatory protein arac, which works with the sugar arabinose to turn on GFP transcription by recruiting RNA polymerase GFP Codes for Green fluorescent protein, which is derived from Aequorea victoria - a bioluminescent jellyfish that fluoresces under UV light. amp r Codes for the enzyme beta-lactamase, which inactivates the ampicillin and allows the cell to grow in the presence of that antibiotic. 2

15 In this lab activity, you will be inserting pglo into non-pathogenic E. coli bacteria. The procedure is never 100% efficient and only a few of your E. coli bacteria will successfully take up the pglo. How will you know which cells contain the plasmid? pglo contains a gene that codes for a protein that protects the cell against the toxic effects of antibiotics. This means that only cells that have the plasmid will survive in the presence of antibiotics. In this procedure, we use ampicillin, an antibiotic very similar to penicillin. This step is called antibiotic selection, and it allows you to select only the cells that have been transformed. The beta-lactamase gene in pglo codes for a protein that breaks down ampicillin. Expression of the beta-lactamase gene in cells that have been successfully transformed allows them to grow in the presence of ampicillin. Non-transformed cells will die or not grow into visible colonies. Your transformed cells will grow on a plate with ampicillin, but they will not fluoresce green until the GFP gene is turned on. Here s where the idea of gene regulation comes into play. Transformed cells will grow on plates not containing arabinose, but will only fluoresce green under UV light when arabinose is included in the nutrient agar. Therefore, arabinose, a sugar that bacteria consume for energy, is the critical ingredient for making your bacteria glow. What s so special about arabinose? It teams up with the arac, the regulatory protein that is expressed by pglo. Regulatory proteins control the timing and location of many cellular processes. Specifically, arac is a transcription factor which, as described previously, functions to turn genes on and off. But it can t turn GFP on by itself it needs the help of arabinose. Together, they work to bring in RNA polymerase, the enzyme that makes RNA, and only then can the glowing, green protein be made. It's a finely orchestrated dance, and all the right players have to be in place for success. Figure 5 Gene regulation of GFP in pglo Figure 5 shows that when arac teams up with arabinose, its shape changes. The protein arac easily forms a bond with the sugar arabinose, and only when they both get close together can the complex function as a transcription factor. What it then does is very simple: it stimulates RNA polymerase to start transcription, and we see firsthand the central dogma of molecular biology in action! The Transformation Procedure In order to increase the chances that your E. coli will incorporate foreign DNA, you will need to alter their cell membranes to make them more permeable. This is a two-step process. First you place your cells and pglo together in a transformation solution (which contains calcium chloride) to neutralize the charge. Second, you quickly heat shock them with a temperature change (42 o C). This hot temperature permeabilizes (loosens) the bacterial cell wall, making it easier for pglo to cross it. This process can be harmful to the cells, so you want to give them a nutritious broth to restart their growth as soon as you re done. Luria Broth (LB) is a liquid that contains proteins, carbohydrates and vitamins so that the E.Coli can rapidly recover and thrive. They will then be placed on an agar medium, a jello-like substance containing LB, with or without antibiotic or sugar, to grow overnight. 3

16 General Lab Skills Required for Success Using Sterile Technique Students should wash their hands before starting lab, after handling recombinant DNA organisms/containers, and before leaving the lab area. All lab surfaces should be decontaminated at least once a day during each class section and following spills. Students should avoid touching the tips of the pipettes or inoculating loops onto any contaminating surfaces, unless instructed in the protocols. Students should practice proper aseptic techniques to prevent contamination. Using Microipettes Students need to be familiar with micropipetting techniques and remember to exchange pipet tips to avoid cross contamination. Do not carry micropipettes sideways or upside down while transferring liquids. Please don t abuse the micropipettors by dialing in amounts beyond their intended calibration limits. When transferring liquids, the student holding the micropipettor should also be holding the microfuge tube of liquid to transfer. Both should be brought to eye level in order to visually confirm that liquid has been transferred. A teammate should confirm the correct micropipettor setting, correct tube of liquid to transfer and the use of clean pipet tips. Success of the lab depends on the proper use of tools and reagents required for the protocol. UV Safety Ultraviolet radiation can cause damage to eyes and skin. Use UV-rated safety glasses or goggles if looking directly at UV light. Using Experimental Controls In this lab, it is important to confirm which cells have received the plasmid, and under which conditions the green fluorescent proteins are being produced. You will need to prepare a series of experimental controls to be able to interpret your results correctly. These controls are designed to minimize the effects of factors other than the single concept that you are testing. Therefore, 2 different reactions will be performed: one with pglo plasmid (+pglo) and one without it (- pglo). See Figure 6 to understand how to set up your reactions. Figure 6: Bacterial growth conditions Nutrient Agar (LB) Antibiotics (ampicillin) On Switch (arabinose) E.Coli + pglo E.Coli -pglo #1 #2 #3 Yes Yes Yes Yes No Yes No The - pglo control serves two roles: 1) to ensure that the bacteria are still alive after the chemical and heat shock procedure, and 2) to make sure that the ampicillin is working property. You will plate these bacteria under conditions #1 and #2, but you should only expect them to grow in condition #1. The +pglo transformation will grow in condition #1 and #2. However, only the bacteria that successfully took up the pglo plasmid will grow in condition #2. In this reaction, you will observe the process of antibiotic selection, but you should not see any GFP produced. Condition #3 is only used for the +pglo reaction. This example proves that the transcriptional control of the GFP gene is intact. The bacteria on this plate are the only ones that should glow green when exposed to UV light. In this reaction, you will observe the process of gene regulation. 4

17 The protocol outlined next describes the procedure for adding plasmid DNA to a bacterial cell. Be sure to follow each step very carefully. You will be cooling your E. Coli cells on ice, then heating them in a water bath, then letting them recover. Make sure your pipetting volumes are accurate at every step. Afterwards, you will grow the cells on a petri dish containing LB agar, antibiotics and arabinose. After 1-2 days, you will look for the development of green fluorescent colonies of bacteria. Student Learning Outcomes at the end of this laboratory, students will be able to: 1. Describe the central dogma of molecular biology. 2. Explain the process of bacterial transformation and selection. 3. Relate the use of bacterial transformation in biotechnology. 4. Differentiate transformed from non-transformed cells. 5. Calculate transformation efficiency and compare with the class data. Preliminary predictions and questions to think about Will the untransformed bacteria appear neon green under a UV lamp? Why or why not? Why don t you attempt to grow the pglo reaction under LB/ara? Do you expect the same number of colonies for the +pglo reaction under condition #1 than condition #2 (on page 4?) Before beginning the transformation, observe a plate of E. coli and a vial of pglo plasmid under a UV lamp. Then view your transformed colonies once you complete the lab activity. Do you see glowing? Fill out the table below: Item Prediction View with UV Lamp Explanation of Results E. coli growing in petri dish on LB agar Vial of pglo plasmid Transformed E. coli grown under condition #2 5

18 Laboratory Activity Place a check mark in the box as you complete each step. 1. Sterilize lab surfaces and wash hands before beginning the lab. 2. Obtain one empty 1.5mL microfuge tubes from your instructor. Using a permanent marker, label the tube +DNA and team intials. **Negative Control: Assigned groups will perform a mock transformation to be used as negative control for the class. Label a second tube DNA. Label each tube twice, on the lid and on the side. Place these tubes into a Styrofoam cup containing crushed ice. pglo Transformation Protocol 3. Add 250µL of Transformation Solution (TS) to each tube using a proper micropipette (Alternatively you can use a plastic transfer pipette) Note: TS contains calcium chloride (CaCl2), which helps neutralize both the bacterial cell wall membrane and DNA charges. Keep your tubes on ice. 4. Obtain a starter plate of E. coli. Observe the colonies growing on it and note what you see. Place the plate on the UV lamp and observe the colonies. Are they glowing? 250µL TS UV Light Wear safety glasses while using the UV lamp. 5. With a sterile inoculation loop, pick up one bacterial colony from the starter plate. Dip and swirl the loop into the +DNA tube to evenly disperse the colony in the solution and release it from the loop. With the cap closed, flick the tube with your finger to mix or rack the tube. Make sure there are no lumps. If doing the negative control, use a new loop to repeat the process for the - DNA tube. Return tubes to ice. 6. Wearing safety glasses, observe the contents of a vial of pglo under a UV lamp. Does it glow? UV Light 6

19 7. With a P-20 micropipettor, transfer 10µL of the pglo plasmid into your tube labeled +DNA only. (Alternatively, use the 10µL inoculation loop. Dip the loop into the 1mL stock plasmid tube. A noticeable film will form around the ring due to surface tension (like a bubble wand). Swirl the loop into tube labelled +DNA.) **DO NOT add plasmid to the DNA tube. Close the cap and flick the tube to mix. 10µL pglo + DNA 8. Incubate both tubes on ice for 10 minutes, making sure the tubes are in contact with the ice. + DNA DNA Incubate 10 minutes on ice 9. While you re waiting, pick up these 3 plates: 1 LB, 1 LB/amp, 1 LB/amp/ara On the outer edge on bottom (non-lid side) of the plate, write +DNA. **If performing the negative control experiment, pick up 1 LB and 1LB/Amp plate. Label them DNA. Also write your team initial or symbol and the date on the bottom (non-lid side) of each plate. PGlo Transformation +DNA LB LB/amp LB/amp/ara *Negative control -DNA LB LB/Amp **only for groups assigned to do the negative control 10. Taking your tubes on ice to the water bath, heat shock your bacteria by transferring both tubes to a foam rack and placing them into a water bath set at 42 C for 50 seconds. Make sure the tubes are pushed down as far as they can go in the rack to contact the hot water. After 50 seconds, quickly place both tubes on ice for another 2 minutes. It is VERY important to watch the time and speed of the transfers. + DNA DNA + DNA DNA Water Bath 42 C / 50 seconds + DNA DNA 2 min 11. Return your tubes to a tube rack now resting on your lab bench. Using a proper micropipette (or transfer pipette), add 250µL of LB broth to each of the tubes 250µL LB Remember to change the tips between the tubes! 7

20 12. Close the tubes. Mix each tube by flicking it several times with your finger. Incubate the tubes for at least minutes at 37 C. You can use the bacterial incubator or other warm place like the top of a refrigerator or keep the tube warm in your hands for this step. This process will allow the transformed bacteria to recover by providing nutrients for their growth. Incubate minutes at 37 C 13. Obtain your labeled plates. Using a P200 (or sterile transfer pipette), transfer 150µL of the +DNA directly to the agar into each plate labeled +DNA plate. Be careful not to poke into the agar! 14. Using a clean inoculation loop, gently spread the liquid on the agar of each plate. You may use the same loop for all the +DNA plates. Be careful not to poke into the agar! Evenly cover as much of the plate as possible. PGlo Transformation +DNA LB LB/amp LB/amp/ara *only for groups doing the neg controls Discard used loops into a waste container with disinfectant. Allow bacteria to soak into the agar plate for a few minutes before the next step. 15. If performing the negative control experiment, repeat step 13 and 14 using the -DNA on the appropriately labeled -DNA plates. Use a clean transfer pipet and inoculation loop for this set. Negative control -DNA LB LB/Amp 16. Invert your plates (lid on bottom). Then stack and tape them together. Make sure to use 2 pieces of tape, one on each side of the stack. Place plates into an incubator oven set at 37 C until the next day or when colonies are visible. Alternatively, stack the plates in a warm spot in the classroom. It may take 2-3 days for bacterial colonies to appear. After the colonies have appeared, you may keep the plates by wrapping them in parafilm and storing in the refrigerator. 17. Clean lab station. Decontaminate all lab surfaces with dilute disinfectant and wash hands following the lab! 8

21 Name Date Period Worksheet: Bacterial Transformation Lab Predictions Will the untransformed bacteria, pglo plasmid, and transformed bacteria all fluoresce green? Before viewing these substances with a UV lamp, list your prediction on whether they will fluoresce green. Then, view them under a UV lamp and provide an explanation of your results. 1. Predictions & Results Item Prediction With UV lamp Explanation E. coli colony No fluorescence No fluorescence Vial of pglo plasmid Transformed E. coli colony No fluorescence Fluorescence No fluorescence Fluorescence Non-transformed bacteria lack the gene for GFP. Plasmid carries gene for GFP but requires bacteria to express gene. Contains plasmids and will fluoresce if arabinose is present. 2. Explain the purpose of these processes or substances during transformation. Process or Purpose Substance a. LB agar Provides nutrients and water needed for bacterial growth. b. Ampicillin or antibiotic c. Calcium chloride d. Heat shock e. Arabinose Prevents growth of untransformed bacteria on LB/amp plates. Allows the DNA to enter the cell. The Ca 2+ neutralizes the membrane s phospholipids and the phosphates within the DNA backbone. Increases the permeability of the cell membrane. Allows the pglo plasmid to enter the cell. Carbohydrate (sugar) responsible for turning on the GFP gene in transformed bacteria. 3. Describe 2 differences and 2 similarities between these bacterial cultures. Condition - pglo DNA bacteria + pglo DNA bacteria Difference Lacks the pglo plasmid. Unable to grow on LB/amp plates. Includes the pglo plasmid. Able to grow on LB/amp plates. Similarity Both will grow on LB agar plates. Both appear white under normal ambient light. 9

22 Name Date Period 4. Before transforming your bacteria, list your predictions below for each of these petri dishes and their contents. Then, describe your results following transformation. Contents LB, -DNA LB/amp, -DNA LB, +DNA LB/amp, +DNA LB/amp/ara, +DNA Predictions* Answers vary Answers vary Answers vary Answers vary Answers vary Illustration of Results Description of Results Bacteria Lawn No growth Bacteria Lawn White colonies Colonies fluoresce under UV light; white colonies if no UV *Possible responses for Predictions - Growth or no growth, fluorescence or no fluorescence under UV light, number of colonies, etc.) 5. Compare your predictions with your actual lab results. Describe how close your predictions were to your actual results and explain possible reasons for any differences. Answers may vary. If responses are different from their results, students may not understand the process and need further review. 6. Explain what may have occurred to produce these results. ( = colony) Contents LB -DNA LB/amp -DNA LB/amp/ara +DNA Illustration of Results Description of Results Possible explanation for results No growth Growth No growth Bacteria not added to plate; too long during heat shock Ampicillin damaged by heat, not added to media or degraded; contamination; mislabeled plate; plasmid was added No plasmid added; plasmid damaged by heat shock; mislabeled plate 10

23 Name Date Period 7. If growth appeared on the LB/amp +DNA plate, would these bacteria a. be transformed? Explain. Yes, in order to grow on the amp plates, these bacteria contain the amp resistance gene gained through successful transformation of the bacteria with pglo plasmid. b. fluoresce under UV light? Why or why not? No. Although the only cells growing on this plate are the ones that took up plasmid and technically they all have the ability to make GFP, they would not be able to express GPF because there is no arabinose in the growth media to bind with arac and turn on the expression of GFP 8. Provide an example of how transformation can be beneficial and an example of how it can be potentially harmful to humans. Condition Transformation example a. Beneficial Answers vary. The production of human insulin and enzymes used for laundry. b. Harmful Answers vary. Inducing antibiotic resistance in undesired organisms. 9. Although transformed cells appear white, with the same phenotypic expression of the wild-type bacteria when the growth media lacks arabinose, they will fluoresce green with a long-wave UV lamp when arabinose is present. Explain why this color change occurs. Under normal light, both sets of colonies appear white even though the GFP is expressed in the transformed bacteria. The GFP gene will only turn on or be expressed in the presence of arabinose due to the plasmid using arac to control expression of GFP. GFP when expressed will only glow green under UV light and not affect how the colonies look under room lighting. 10. Provide a rational or benefit of adding DNA sequences coding for fluorescent proteins such as GFP, to tag genes of interest in plasmids used for transformation. Adding a gene for a fluorescent protein along with the desired gene will help flag a successful transformed cell easily through visual inspection under UV light. You can also tag proteins to learn more about the location of the protein or its function. 11. In your own words, explain the process of transformation. Answers may vary. Transformation is a process in which additional DNA is added into a cell, usually with a plasmid, to produce new, desirable and beneficial proteins. Transformed bacteria will then produce new traits not found in the original bacteria. 11

24 Worksheet: Calculating Transformation Efficiency When performing transformation experiments, you usually want to obtain as many transformants as possible. This is important because you want to make sure your conditions for transformation is at its optimum. Transformation efficiency is the efficiency whereby cells take up the introduced DNA. Many factors contribute to transformation efficiency: cell age and competency (the ability to take up DNA), the type of cells being transformed, plasmid length and quality, the method of transformation (heat shock or electroporation) and just different conditions in general. Having a low transformation efficiency may point to poorly competent cells, poor conditions, or poor techniques (not following protocol). In a research lab, it s good to have many transformants for research, just in case individual transformants may not work as well (e.g. different levels of expression), or some other unknown problems associated with transformed cells. In making a genomic library, you want as many transformants as possible to have a robust library. In cell culture, you may take a population of transformed cells for further study therefore having a high transformation efficiency allows for better study. In this exercise, we will calculate the transformation efficiency of the E. coli bacteria by pglo. The data can then be gathered from each team of the class and the data compared with a different transformation technique called electroporation. Transformation efficiency calculation: The number of colonies observed growing on an agar plate (cfu) Amount of DNA used (in µg) cfu=colony forming units Two data are needed for this: 1. Total number of green fluorescent colonies on your LB/amp/ara plate. 2. Total amount of pglo plasmid DNA used for bacterial transformation that was spread on the LB/amp/ara plate. 1. Determine the total number of transformed green fluorescent colonies. Place the LB/amp/ara plate near a UV light source. Count the number of green fluorescent colonies that glow under UV light. Enter that number here Total number of colonies = 2. Determine the amount of pglo DNA in the cells spread on the LB/AMP/Ara plate. Two pieces of information are needed: a) The total amount of DNA you used for the +DNA in the experiment b) The fraction of DNA that was spread onto the LB/amp/ara plate a. Total amount of DNA: DNA in µg = (concentration of DNA in µg/µl) x (volume of DNA in µl) In this experiment, 10µl of pglo at a concentration of 0.01 µg /µl was used. Enter that number here Total amount of pglo DNA, µg used in this experiment = 12

25 b. Fraction of pglo plasmid DNA (in the bacteria). For this experiment, a certain amount was spread onto each plate. To find that fraction: Fraction of DNA used Sample volume spread on LB/amp/ara plate, in µl Total sample volume in tube, in µl 150µl of cells was spread from the tube containing a total volume of 500µl of solution. Enter that number here Fraction of DNA= c. How many µg of pglo DNA was spread on the LB/amp/ara plate? Multiply the total amount of pglo DNA used by the fraction of pglo DNA you spread on the LB/amp/ara plate. pglo DNA spread (µg) = amount of DNA used (µg) x fraction of DNA Enter that number here pglo DNA spread, µg = Now, we are finally ready to calculate the transformation efficiency! Number of colonies on LB/amp/ara plate = pglo DNA spread, µg = Transformation efficiency calculation: The number of colonies observed growing on an agar plate Amount of DNA used (in µg) Enter that number here Transformation efficiency = transformants or cfu/µg cfu=colony forming units 13

26 Analysis of results: What is the transformation efficiency of each team in the class? Team Efficiency Do you see differences between the teams? Why do you think there are these differences? What could increase/decrease transformation efficiency? In past studies, this method of heat shock protocol that was performed by research labs usually has a transformation efficiency between 8x10 2 and 7x10 3 transformants per microgram of DNA. How does your team s result compare to this data? How does the class result compare to your data and to the data by research labs? Another method for transformation is called electroporation. In this method, an electric field is applied to allow the cell membrane to open up and take up DNA. The transformation efficiency from electroporation may be 1x10 8 cfu/μg. What fold higher is the transformation efficiency by electroporation vs. heat shock? 14

27 BABEC Educational Transformation Kits BABEC thanks Qiagen for their generous support of plasmid prep kits for BABEC bacterial transformation labs. Acknowledgements The following images have been provided courtesy of: Figure 1 adapted from National Geographic. Figure 2 Tsien Laboratory at UCSD. Figure 3 Wikipedia. Figure 4 adapted from Bio-Rad. 15