pGLO Bacterial Transformation Kit Instructor Guide PDF

BIO-RAD pGLO Bacterial Transformation Kit for General Biology Catalog #17006991EDU...

BIO-RAD pGLO Bacterial Transformation Kit for General Biology Catalog #17006991EDU Instructor Guide Note: Duplication of any part of this document is permitted for noncommercial, educational use only. Commercial use of copyrighted content, including use in instructional materials for which payment is received, requires express permission from Bio-Rad Laboratories, Inc. Contact us at [email protected] for more information. For technical support, call your local Bio-Rad office, or in the U.S. call 1-800-4BIORAD (1-800-424-6723) option 2. Dear Instructor A brilliant flash of glowing green has the power to spark excitement and a sense of wonder. In these activities, your students will genetically engineer their own green glowing bacteria with a gene borrowed from Aequorea victoria, a bioluminescent jellyfish. It was such a transference of genetic information that ignited an era of unprecedented advancement in life science and has become a cornerstone of biotechnology. As your students discover how the vast diversity of life relies on a shared four-letter code and make sense of the tension and dramatic inner workings of a cell, they participate in a longstanding scientific conversation. The lessons in the kit are designed for your students to make sense of gene expression and bacterial transformation through progressive rounds of careful observation and data analysis. They will be asked to describe their thinking and to justify their claims using evidence, each time with more complex and nuanced explanations. The experience culminates in an engineering design challenge in which your students apply their new skills in bacterial transformation to address a real world problem. The activities in this kit were developed in partnership with Elizabeth Martinez, an esteemed curriculum developer for the Illinois Mathematics and Science Academy in Aurora, Illinois. We strive to continually improve our curriculum and products, and your input is extremely important to us. We welcome your stories, comments, and suggestions. Share your students’ success on social media @bioradeducation. Bio-Rad Explorer Team Bio-Rad Laboratories 6000 James Watson Drive, Hercules, CA 94547 [email protected] i Table of Contents Before You Start Kit Storage................................................................................................................................................. 1 Safety......................................................................................................................................................... 1 Kit Components......................................................................................................................................... 2 Ordering Information................................................................................................................................... 3 Kit Activity Overview................................................................................................................................... 4 Activity Timelines........................................................................................................................................ 5 Curriculum Fit............................................................................................................................................. 6 Instructor Preparation Preparation Instructions.............................................................................................................................. 7 Instructor Background.............................................................................................................................. 12 Instructor Guide Activity 1: Transferring Genes between Species....................................................................................... 15 Activity 2: Bacterial Transformation Laboratory Activity............................................................................. 16 Activity 3: Bacterial Transformation Design Challenge............................................................................... 17 Appendix A: Small Microwave Modified LB Agar Plate Preparation Instructions........................................ 18 Resources................................................................................................................................................ 20 Legal Notices........................................................................................................................................... 20 explorer.bio-rad.com ii  Before You Start 00:00 Kit Storage Safety Guidelines LB ag LB LB/amp LB/amp When you receive the pGLO Bacterial Transformation Kit for General Biology: Basic laboratory safety guidelines should be followed at all times: wear gloves and safety goggles; eating, drinking, smoking, and applying cosmetics are not permitted in the work area. 1 Record storage location and batch numbers from the product labels. 250 µl The Escherichia coli bacteria HB101 K-12 strain contained in this kit is nonpathogenic and has 2 Store the Transformation Reagent Refill in the 4°C been genetically modified to prevent its growth refrigerator (4°C). unless grown on an enriched medium. However, E. coli LB agar LB broth LB/amp handling of the E. coli K-12 strain Lyophilized requires the E. coli 500 ml LB/amp use of standard Microbiological Practices. These –20°C 3 Store the remaining materials at room E. co Room Temp practices include, but are not limited to, the ~20°C temperature. following guidelines. Decontaminate work surfaces E. c once a day and after any spill of viable material. Decontaminate all contaminated liquid or solid 4 Visit bio-rad.com/pgloGenBio to download wastes before disposal. All persons must wash the most up-to-date instructor and student guides. their hands: (i) after they handle material containing bacteria, and (ii) before exiting the laboratory. Perform all procedures carefully to minimize the RNA polym creation of aerosols. After the lab activities, araC place all bacteria plates and any materials that may have contacted bacteria Ampic illin 250 µl 3 ml in a 10% bleach solution Ampicillinfor at least 20 minutes. AraC C ori 5.75 ml pGLO Follow local regulationsPBADfor further disposal recommendations. Transformation Transformation Solution Solution Transformation Promoter Solution E. coli Ampicillin GFP Transformation Arabinose Transformation Ampicillin Ampr may cause allergic reactions or irritation solution Transformation solution AraC RNA polym po y Lyophilized pGLO plasmid DNA solution to the eyes, respiratory system, and skin. In case Ampicillin Technical Support is available at [email protected] Arabinose or of contact with eyes, rinse immediately with plenty Arabinose 1-800-4BIORAD, option 2. of water and seek medical advice. Wear suitable protective clothing. Ampicillin is a member of the Promoter penicillin family of antibiotics. Those with allergies to penicillin or to any other member of the penicillin family of antibiotics should avoid contact with ampicillin. Ag LB ar 8g 12 g 250 µl 0.5 ml Transformation Solution Transfo Solu TS LB 00:00 Transformation LB broth LB LB/amp Transfo solution solu LB LB/amp 1  Before You Start Kit Components Each kit contains materials for 12 student workstations. Item Quantity Transformation solution 15 ml E. coli HB101 K-12, lyophilized 1 vial pGLO plasmid, lyophilized 20 µg Ampicillin, lyophilized 20 mg L(+) arabinose, lyophilized 600 mg LB nutrient broth, sterile 10 ml LB nutrient agar powder, sterile 20 g Plastic transfer pipet, sterile 50 Inoculation loop, sterile, 10 µl 80 pGLO Bacterial Transformation Kit for General Biology Petri dish, 60 mm, sterile 40 Multicolor microcentrifuge tube, 2.0 ml 60 Foam micro test tube holder 8 UV pen light 1 Required Materials (not included in this kit) Quantity Microwave oven 1 Temperature-controlled dry bath or water bath 1 Thermometer (0–60°C) 1 Erlenmeyer flask, 1 L 1* Graduated cylinder, 500 ml 1 Distilled water 500 ml Ice bath (e.g., ice bucket or foam cup) 12 Marking pen 12 Timer to count seconds Laboratory tape Household bleach, 10% solution for cleanup Recommended Materials (not included in this kit) Quantity 2–20 µl adjustable-volume micropipet and tips 1 Incubator oven, 37°C 1 Vortexer 1 Tube rack 1 Parafilm laboratory sealing film * If your microwave cannot accommodate a 1 L flask, see Appendix A for modified LB agar plate preparation instructions. You will need two 500 ml flasks instead of a single 1 L flask and a balance with at least 10 g capacity. explorer.bio-rad.com 2 Before You Start Ordering Information Catalog # Description Kits and Refill Packs 17006991EDU pGLO Bacterial Transformation Kit for General Biology 1660555EDU Transformation Reagent Refill 1660005EDU Green Fluorescent Protein Chromatography Kit 1660013EDU pGLO Kit SDS-PAGE Extension Consumables 1660405EDU pGLO Plasmid, lyophilized, 20 µg 1660406EDU Arabinose, lyophilized, 600 mg 1660407EDU Ampicillin, lyophilized, 30 mg 1660408EDU E. coli Strain HB101 K-12, lyophilized 1660409EDU Transformation Solution, 15 ml 1660421EDU LB Broth, 10 ml 1660600EDU LB Nutrient Agar Powder, 20 g, makes forty 60 mm agar plates 1660472EDU LB Nutrient Agar Powder, 500 g, makes one thousand 60 mm agar plates 1660469EDU Petri Dishes, 60 mm, sterile, pack of 20 1660470EDU Petri Dishes, 60 mm, sterile, pack of 500 1660471EDU InoculationLoops, sterile, pack of 100 1660473EDU Colored 1.5 ml Microcentrifuge Tubes, 6 colors, pack of 600 1660474EDU Disposable Plastic Transfer Pipets, sterile, pack of 500 2239430EDU 2 ml EZ Micro Test Tubes, natural, pack of 500 Equipment and Laboratory Supplies 1660501EDU Mini Incubation Oven, 120 V 1660610EDU BR-2000 Vortexer, 120 V 1660611EDU BR-2000 Vortexer, 220 V for Europe 1660621EDU BR-2000 Vortexer, 220 V for the UK 1660500EDU Long-Wave UV lamp 1660530EDU Long-Wave UV Pen Light 1660481EDU Green Racks, set of 5 racks 1660479EDU Jellyfish Foam Floating Racks, 12-wells, pack of 8 3  Before You Start Kit Activity Overview Activity 1 Transferring Genes between Species Students observe the growth of green fluorescent bacteria. They assemble ➜ descriptive models from their observations and make predictions about how antibiotic resistance might impact growth. Activity 2 Bacterial Transformation Laboratory Activity Students transform bacteria with an engineered plasmid without ➜ arabinose. Then they design an experiment to use arabinose to turn on the gene for green fluorescent protein. Activity 3 Bacterial Transformation Design Challenge (optional) + + + = + + + = Students use their new understanding of bacterial transformation to design a plasmid-based biosensor that can be used to solve real world ➜ + + + = problems. explorer.bio-rad.com 4 Before You Start Activity Timelines The activities in this kit are designed to take five 50 min class periods as shown in Table 1. Table 1. Suggested timeline for daily 50 min class periods. Class Period 1 Class Period 2 Class Period 3 Class Period 4 Class Period 5 Activity 1 Activity 2 Activity 2 Activity 2 Activity 3 (optional) Part 1. Part 1. Analyze results from Analyze results from Continue work and Observe Fluorescent Transform Bacteria Part 1 Part 2 share out Organisms with pGLO Plasmid Part 2. Activity 3 Part 2. Incubate plates Switch on the GFP (optional) Bacterial Model the Processes overnight at 37°C. gene Transformation Design that Occur in Green Challenge Fluorescent Bacteria Students conduct their experiments Part 3. and incubate plates Analyze the pGLO overnight at 37°C. Plasmid Finish group work out of class or spend an extra day for Activity 1. 5  Before You Start Curriculum Fit Required prior knowledge Basic DNA, RNA, and protein structure and function Central dogma (DNA → RNA → Protein → Trait) — the provided lesson sequence requires that students have a basic understanding of the central dogma. If students are not yet familiar with central dogma, you can use the phenomenon in Activity 1, Part 1 to get students engaged in asking questions that may lead to further instruction How bacteria grow and divide How to culture bacteria on solid media How to use a pipet Concepts, topics, and skills Gene expression and regulation — students will compare the transcription and gene expression of Ampr, the gene whose product confers resistance to ampicillin, and GFP, which codes for green fluorescent protein (GFP) Bacterial transformation — students will perform a bacterial transformation and apply what they learn to design their own plasmid-based biosensor Genetic engineering — manipulating or engineering the genetic information of an organism to change phenotype is the goal of genetic engineering. Students will introduce a gene naturally found in jellyfish into bacteria using bacterial transformation Artificial selection — after students complete the transformation, they will be confronted with evidence of artificial selection. Bacteria that take up the pGLO plasmid, which includes an ampicillin resistance gene, will grow in the presence of ampicillin, while those that do not take up the plasmid will not grow Biosensors — students have the opportunity to apply their new knowledge toward the design of a biosensor, an engineered biological system that responds to an input explorer.bio-rad.com 6  Instructor Preparation Preparation Instructions Preparation step Time required When to begin preparation Prepare LB agar plates 1–2 hr plus 2 days to dry plates 3–28 days before Activity 2 Rehydrate E. coli 5 min plus 8–24 hr incubation at 37°C 2 days before the activity Streak starter plates 20 min plus 24 hr incubation at 37°C 24 hr before the activity Dispense solutions 30 min Up to 3 days before the activity Tips If you do not have an incubation oven, incubate rehydrated E. coli and starter plates at room temperature (20–25°C) for 72 hr total. Incubating at room temperature for 72 hr may help coordinate preparation over a weekend Visit bio-rad.com/pglo for tutorial videos that explain the preparation techniques used Prepare LB Agar Plates 3–28 days before Activity 2 If your microwave cannot accommodate a 1 L flask, see Appendix A for 500 ml gar –20°C modified plate pouring instructions. Room Temp ~20°C 1. Add 500 ml distilled water to an empty 1 L flask. ➜ Ag LB 500 ml LB –20°C ar Room Temp ~20°C Ampic 2. Add 20 g LB agar powder to the flask. Swirl to mix. ➜ illin 3 ml Ampicillin Transformation Solution LB/amp Ampicillin Transformation Ag 500 ml LB solution ar Ampicillin 00:00 3. Heat the flask to boiling in a microwave, about 3 min. Swirl and ➜ Ampic illin repeat 3boiling ml at least twice more until all the agar has dissolved Ampicillin (no more clear specks). Transformation Solution LB agar LB/amp Safety! Use a hot pad or mitt when handling the hot flask. Be careful LB/amp Ampicillin not Transformation to allow the LB agar to boil over. Use a lower power microwave LB/amp solution setting and watch it carefully. Always allow the molten agar to cool Ampicillin slightly (about 30 sec) before swirling to prevent sudden boil over. Ampic 250 µl 0.5 ml illin Ampicillin 7 Transformation Solution 4°C Transformation Solution TS LB LB agar LB br LB/amp  Instructor Preparation 4. Label 26 plates LB and 14 plates LB/amp. ➜ Ag LB ar Note: Label the plates on the bottom, close to the edge with a permanent marker. Do not label the lids. 00:00 Note: This protocol produces two extra LB and two extra LB/amp plates which can be used as replacements or for further inquiry. LB LB/amp LB/amp –20°C Room Temp ~20°C 00:00 250 µl 5. Fill 26 LB plates one-third to one-half full (~10 ml) with molten ➜ LB agar. Replace the lids immediately after pouring the agar. 4°C E. coli LB LB/amp LB agar LB agar LB broth LB Ag LB/amp 500 ml LB –20°C ar Lyophilized E. coli LB LB/amp Room Temp ~20°C Ampic illin 250 µl 3 ml Ampicillin A. 6. Add 3.0 ml transformation solution to the vial of ampicillin. Vortex or ➜ Transformation LB/amp 4°C mix by pipetting until dissolved. Solution E. coli LB broth Ampicillin Transformation Lyophilized E. coli B. Note: Transformation solution is used because it is a convenient solution RN Ampicillin sterile solution. If you have sterile water, that may be used instead. E. coli Ag 500 ml LB –20°C ar E. coli araC C. Ampic mp illin C 3 ml Ampicillin 250 µl AraC C 00: ori 5.75 ml Transformation pGLO Solution PBAD Transformation 7. Once the flask has cooled enough that it can be comfortably held ➜ D. Solution Transformation Pro (~50°C), add the rehydrated ampicillin. Gently swirl to mix. Solution Ampicillin E. coli Transformation GFP solution nsformation Amp Arabinose solution Note: Lyophilized pGLO Excessive heat (>60°C) will destroy ampicillin. BeAmpicillin Transformation sure not to r LB/amp RNA polymerase AraC R solution plasmid DNA add it until the agar has cooled enough to Arabinose handle. However, the agar Arabinose will solidify at 27°C so be sure to pour your plates before it has cooled araC too much. Placing your flask in a 50°C water bath will help prevent 250 µl Pro the agar from cooling too quickly. AraC C ori Transformation pGLO Solution Ampic PBAD illin TS 3 ml Ampicillin 8. Fill 14 plates one-third to one-half full (~10 ml) with molten LB/amp ➜ Promoter GFP GFP Transformation solution LB broth 4° agar. Ag LB ar Ampr LB agar rabinose AraC RNA polymerase po y e ase LB/amp Ampicillin 8g tion 12 g Arabinose LB/amp binose Direction of transcription Ampicillin 250 µl 0.5 ml 25 Promoter GFP Transformation Solution Transformation 8 Solution explorer.bio-rad.com TS LB 00:00 Transformation LB broth solution Transformation solution  Instructor Preparation LB LB/amp 9. Once solidified (~30 min), allow the plates to dry unwrapped at room Ag LB ar temperature (20–25°C) for two days. Note: Allowing plates to dry for two days improves the uptake 00:00 of the liquid transformation samples in the student lesson. Ampic illin in 10. Stack and wrap plates in plastic or in the original plastic sleeve ➜ 4°C packaging taped closed. Store plates upside down and refrigerated LB LB/amp LB/amp at 4°C. Plates can be stored this way forLB upagar to one month before use. LB/amp LB broth LB/amp Rehydrate bacteria 2 days before Activity 2 If you will be incubating rehydrated E. coli and starter plates at room temperature, rehydrate bacteria 4 days before Activity 2. 00:00 250 µl 11. Using a new pipet, add 250 µl LB broth to the vial of E. coli. Recap ➜ the vial and shake gently to resuspend the bacteria. 4°C E. coli LB agar LB agar LB broth LB LB/amp LB/amp 12. Incubate the vial 8–24 hr at 37°C. LB Lyophilized E. coli LB/amp LB araC 0.5 ml 250 µl 250 µl ori 5.75 ml A. pGLO Streak starter plates 1 day before Activity 2 PB 4°C Transformation Solution If you will be incubating the starter plates at room temperature, streak Transformation Solution LB E. coli GFP LB broth plates three days before Activity LB broth2. E. coli Transformation Lyophilized E. coli Arabinose B. Ampr solution Transformation Lyophilized pGLO solution 13. Using a new inoculation loop, streakplasmid the rehydrated DNA E. coli onto ➜ E. coli Arabinose 12 LB plates. araC E. coli C. Important! Follow the streaking pattern shown in steps A–D. Dip the loop ori 250 µl AraC C 5.75 ml into the rehydrated E. coli, then streak the plate side to side at the edge pGLO PBAD Transformation Solution of one quadrant (A). Rotate the plate about a quarter turn. Pass the loop Transformation Solution D. from side to side through the previous streaks multiple times, extending E. coli GFP Transformation into the next quadrant (B). Repeat twice more (C, D). Arabinose Ampr Ag solution Transformation LB AraC ar Lyophilized pGLO solution plasmid DNA RNA polymerase Arabinose 8 g Arabinose 14. Incubate starter plates upside down in a 37°C incubator oven for 12 g 24 hr. Do not refrigerate before use. araC 200 ml Note: Alternatively, starter plates may be incubated upside down at AraC C room ori temperature (20–25°C) for 72 hr. pGLO PBAD 00:00 Promoter GFP GFP LB LB/amp Ag LB ar Ampr Arabinose 8g AraC RNA polymerase po y e ase 12 g B abinose LB/amp Arabinose Direction of transcription Promoter GFP 9 00:00 LB LB/amp  Transformation Solution Instructor Preparation LB/amp Ampicillin Transformation solution Dispense solutions up to 3 days before Activity 2 Ampicillin Ag 500 ml LB 20°C ar 15. Add 250 µl transformation solution to each of 24 tubes labeled TS. ➜ 250 µl Ampic illin 00:00 3 ml Ampicillin Transformation Solution Transformation TS Solution LB agar LB/ Transformation LB broth Ampicillin solution Transformation LB/amp LB/amp solution Ampicillin 250 µl 0.5 ml Ampic Transformation 16. Add 0.5 ml LB broth to each of 12 tubes labeled LB. Solution ➜ Transformation illin Solution Ampicillin TS LB Transformation LB broth 4°C solution Transformation solution LB agar LB/amp LB/amp 200 ml 250 µl 0.5 ml 300 ml 250 µl Transformation 17. Add 250 µl transformation solution to the pGLO plasmid vial. ➜ Solution Transformation Solution Transformation Solution TS LB E. coli Transformation LB broth Transformation Distilled water solution solution Transforma Lyophilized pGLO solution plasmid DNA LB 200 ml 300 ml 0.5 ml 250 µl 5.75 ml ori Transformation Solution Ag Distilled water 18. Add the remaining 5.75 ml of transformation solution to the vial of ➜ Transformation LB ar Solution Ampic LB illin arabinose. Vortex or mix by pipetting for about 30 sec to begin E. coli LB broth 8g Ampicillin Transformation Am