RECOMBINANT DNA: DUAL ANTIBIOTIC-RESISTANCE GENES Prepared by the Office of Biotechnology, Iowa State University DRAFT 4/03 ** Portions of this protocol were adapted from DNA Science: A First Course in Recombinant DNA Technology. Cold Springs Harbor Laboratory Press and Carolina Biological Supply Company PRE-LAB INFORMATION: IMPORTANT----PLAN AHEAD! The teacher(s) need to prepare materials several days before the actual lab. Preparing the media for the transformation part of the lab will take longer than the regular transformation. You will be preparing four different kinds of media plates for transformation in advance of the lab. (See transformation prep instructions.) Timing the growth of the bacteria to be used in the transformation is critical. You will need to grow the bacteria to a mid-log phase in an LB broth culture, ideally 3-4 hrs before proceeding to the Competent Cell Procedure (Day 2). You should plan six uninterrupted class periods of 45-50 minutes each to complete the exercise. Class periods do not have to be consecutive as long as solutions can be stored in the refrigerator or freezer. A good rule to follow is after a heating, you can hold a solution overnight in the refrigerator unless otherwise indicated by the procedure. TEACHER INFORMATION: The process of constructing and analyzing recombinant molecules in this laboratory requires participants to follow directions carefully. The protocol has been optimized to fit into a 45- minute classroom session. The teacher should plan well ahead of the laboratory day to ensure good timing of events that are critical for successful lab results. The starting parent plasmids are pamp and pkan, each of which carries a single antibioticresistance gene ampicillin resistance in pamp and kanamycin resistance in pkan. The goal is to construct a recombinant plasmid that contains both ampicillin and kanamycin resistance genes. The transformed bacteria are selected on Luria broth agar containing both ampicillin and kanamycin antibiotics. In Day 1 of the student lab, the plasmids pamp and pkan are digested in separate reactions with enzymes BamHI and HindIII. Each plasmid contains a single recognition site for each enzyme, so each plasmid digest yields only two restriction fragments. The pamp digest yields fragments of 3755 base pairs (bp) and 784 bp, while the pkan digest yields fragments of 2332 bp and 1861 1
bp. After the digests are complete, the reactants can be stored at -20 o C for several days before proceeding to the Ligation part (Day 3) of the student lab. In Day 3 of the student lab, the four fragments from the digests are ligated together to form recombinant molecules. It should be noted that three viable combinations of recombinant plasmids can be produced: (1) the dual resistance plasmid containing Amp/Kan genes and the two single resistance plasmids, (2) a plasmid containing the Amp gene and (3) a plasmid containing the Kan gene. The digested plasmids of pamp and pkan are mixed together with DNA ligase, ATP, and Mg ++ and incubated at room temperature. The sticky ends of BamHI and HindIII fragments will complementarily realign themselves, producing three viable plasmids: pamp/kan, pamp, and pkan. Ligase catalyzes the formation of covalent phosphodiester bonds that link the complementary ends into stable recombinant DNA molecules. TEACHER PREPARATION FOR RECOMBINANT DNA TRANSFORMATION Preparation for the DNA transformation part of this exercise should begin at least 2-3 days in advance of the laboratory period. Luria Broth (LB) and LB plates can be purchased from several supply companies either as pre-pour plates or pre-made, ready-to-use media. The following supplies should be provided to each group of three students: * 1-1.5 ml microcentrifuge tube * 1 ml of sterile CaCl 2 labeled "CaCl 2." Store on ice until needed. * 1 aluminum foil packet containing 6 sterile toothpicks or sterile inoculating loops * 1 aluminum foil packet containing the glass part of 6 sterile pasteur pipettes or sterile 5ml graduated plastic transfer pipettes. * 1 rubber bulb (if using glass pasteur pipettes) * 1 aluminum foil packet containing 6 sterile paper clips that are large and smooth. The clips should be opened into a 90 o angle and the small loop of the clip bent straight. The large looped end of the clip will act as a bacterial spreader. * 1 Sharpie marking pen * 1 glass test tube containing 2 ml of sterile Luria broth and labeled "LB" * 2 petri dishes containing LB agar labeled "LB No Amp/Kan" on the bottom. One will be used for the fresh bacteria culture used to make mid-log suspension. * 1 petri dish containing LB agar and the antibiotic ampicillin. The dish should be labeled "LB Amp" on the bottom. * 1 petri dish containing LB agar and the antibiotic kanamycin. The dish should be labeled "LB Kan" on the bottom. * 1 petri dish containing LB agar and both antibiotics ampicillin and kanamycin. The dish should be labeled "LB Amp/Kan" on the bottom. * 3 copies of the laboratory instructions, one for each student 2
The following supplies can be shared by 6 students: * 1 petri dish containing colonies of MM 294E. coli (See section on Bacterial Culture Preparation, streak plating.) * 1 container for used materials such as toothpicks, pipette tips, etc. The teacher should have available for the entire class: * 1 incubator for the petri dishes set at 37 o C or less. It is difficult to maintain the temperature precisely unless a research incubator is used. Prolonged temperatures above 40 o C will kill the bacteria. Temperatures lower than 37 o C will result in slower growth of the bacteria, but will not kill them. * 1 Sharpie marking pen * Containers for placing tubes on ice after ligated DNA has been added, such as a Styrofoam cup or Thermos cooler * Containers for the 37 o C, 42 o C, and 65 o C water baths, such as a Styrofoam cup or a second water bath set at 42 o C and 65 o C * Microcentrifuge (MicroV from Office of Biotechnology) STERILIZATION OF SUPPLIES 1. Sterilization of packets of toothpicks, glass pipettes (if appropriate), and paper clips can be accomplished by wrapping each item in aluminum foil, labeling the contents with a marking pen, and (a) baking them in an oven at 350 o F for 15 minutes, or (b) putting them in a pressure cooker at 15 pounds for 15 minutes, or (c) placing them in an autoclave for 15 minutes. The pressure cooker and autoclave should be at the desired pressure for the 15 minute period. After the packets have cooled, they should be stored unopened at room temperature. The students should be instructed when opening the packets to touch only that part of the object that will not come in contact with the solutions or petri dishes. 2. Sterilization of the 1.5 ml microcentrifuge tubes can be accomplished by wrapping in aluminum foil all of the tubes needed by the teacher to prepare the supplies for the students. The tubes can be: (a) baked at 250 o F for 30 minutes (they melt at 350 o F), or (b) put in a pressure cooker at 15 pounds for 15 minutes, or (c) placed in an autoclave for 15 minutes. 3. Calcium chloride. Dissolve 0.75 g of CaCl 2 in 50 ml of distilled water in a labeled 100-ml glass bottle with a cap. Twist the cap snug and place it in: (a) boiling water for 30 minutes, or (b) a pressure cooker at 15 pounds for 15 minutes, or (c) an autoclave for 15 minutes. 3
Allow the bottle to cool until it is comfortable to hold, cap it tightly, and store in a refrigerator until used. 4. Ampicillin solution. Make the 5% solution by adding 1 ml of sterile, cool distilled water to the 0.05 g of ampicillin salt provided by the ISU Office of Biotechnology. For each 100 ml of LB Amp agar to be prepared, add 2 drops of ampicillin solution. If you use a commercially prepared 1% solution of ampicillin, add 1 ml of ampicillin solution to each 100 ml of LB Amp agar. The water can be sterilized by placing it in a glass bottle that is not more than half full, putting the cap on snug, and using methods a,b,or c described in Step 3 for the calcium chloride. The sterile water should be stored in the refrigerator until it is used to make the ampicillin solution. The ampicillin solution should not be prepared and stored in advance for an extended period. The solution should be prepared and put in the refrigerator immediately after the agar plate solutions (Item 7) are prepared. 5. Kanamycin solution. Repeat step 4 using kanamycin antibiotic instead of ampicillin. 6. Luria broth (LB) solution. Calculate the amount of nutrient broth that will be supplied to the students and add extra for spillage and other factors. Weigh 25 mg of LB premix /ml (25g/liter) of distilled water into a bottle and label it. Add the appropriate volume of distilled water to the bottle. Microwave the solution to make sure all the LB is dissolved. Prepare one glass test tube of LB for each group. Place 2 ml of the LB into the glass test tubes, tighten the caps, and place them in an appropriate rack in boiling water for 30 minutes to sterilize them. After the 30 minute-period, remove the tube rack from the boiling water and let the tubes cool. Unused broth can be reboiled and stored in the refrigerator for future use. MAKING LB PLATES 7. If using the boiling water bath method (3a), it is best to dissolve the agar and nutrient broth powders by heating the suspension in a microwave before sterilizing the solution. To dissolve the agar mixture, put on a heat-resistant glove. With the cap loose, microwave the suspension until it boils, swirl the bottle(s), and alternate boiling and swirling until there are no visible solids. Then place the bottle in a boiling water bath and sterilize according to Item 3a. After sterilizing the agars mixture, the bottle(s) should be swirled to mix the solution and cooled at room temperature to 55 o C, at which point the bottle(s) can be held without an insulated glove. The petri dishes labeled "LB No Amp/Kan" should be poured immediately. The bottom of the dish should be covered with the agar. Agar begins to solidify at about 45 o C. Therefore, it is important to pour the plates as rapidly as possible. If the "LB No Amp/Kan" agar does solidify, it can be reboiled 4
and used again. Rinse the bottle with a large amount of tap water immediately after use so that the agar does not solidify in the bottle or in the sink. LB agars that contain antibiotics should be prepared and poured when the temperature reaches 55 o C. Add the antibiotic and pour immediately into the plates. Luria Broth Agar plates: You will make four types of agar plates, (1) without ampicillin or kanamycin "LB No Amp/Kan, (2) with ampicillin, LB Amp, (3) with kanamycin LB Kan, and (4) with both ampicillin and kanamycin LB Amp/Kan. For each plate, 25 ml of agar solution will be required. Label each plate on the underside, not the lid, before it is poured. LB No Amp/Kan plates: Prepare two "LB No Amp/Kan plates for each group of 3 students. One will be used for a starter culture, the other as a control for the lab. It is best to prepare a few extra plates for the entire class in case contamination occurs in one or more of them. Place the required volume of distilled water in one or more glass bottles with caps. The bottles should not be more than half full. Add 25 mg of LB premix and 15 mg of agar /ml (25 g of LB and 15 g of agar per liter) of distilled water. With the caps loose, sterilize the solution by one of the methods described for the calcium chloride (Step 3a, b, or c). "LB Amp" plates: Prepare 1 "LB Amp" plate for each group of 3 students. Follow the same procedure as for the " LB No Amp" plates until the agar has cooled to 55 o C. Add 2 drops of the ampicillin solution (Step 4) per 100 ml of solution, swirl to mix, and pour immediately into the plates labeled "LB Amp". If the agar solidifies, it cannot be reheated because the ampicillin will be destroyed above 60 o C. LB Kan plates: Prepare 1 LB Kan plate for each group of 3 students. Follow the same procedure as for the "LB Amp" plates. LB Amp/Kan plates: Prepare 1 LB Amp/Kan plate for each group of 3 students. The procedure is the same as for the "LB Amp" and LB Kan plates, except to the agar you will add 2 drops of ampicillin and 2 drops of kanamycin solution per 100 ml of LB agar. Allow the "LB No Amp/Kan," "LB Amp," LB Kan, and LB Amp/Kan plates to harden and dry overnight, and then turn the dishes upside down (lid down, agar up). If they are to be kept for more than 2 days, parafilm the dishes or place them in a plastic bag, and store them upside down in a refrigerator. The plates can be kept refrigerated for a month. Note: People differ in their sensitivity to temperature and a teacher may prefer to measure the temperature of the agar to determine when 55 o C is reached, particularly for the solution to which ampicillin is added. It is not possible to put a thermometer 5
into the heated agar solution because it will become contaminated. There are two alternatives that can be used. (A) The bottle of agar can be put into a container with the same volume of cool tap water as the volume of the medium inside the bottle. When the temperature of the tap water reaches 55 o C, the contents inside the bottle should be at a similar temperature. (B) The bottle of agar can be put into a hot water bath at 55 o C and allowed to stand for 30 minutes. BACTERIAL CULTURE PREPARATION: Streak Plating: One day before Competent Cell procedure (Day 2 of the student lab), the teacher should prepare a streak plate of E.coli bacteria to be used for transformation. Use a sterilized transfer loop, a paper clip bent into a loop and sterilized, or a sterilized toothpick. Use the device to touch a colony of bacteria from a petri dish or test tube. Spread the bacteria on the plate in a zig-zag pattern to obtain individual colonies as the concentration of bacteria on the transfer device becomes less. The best results can be achieved by streaking an LB plate with bacteria and incubating at 37 o C overnight before doing a mid-log suspension procedure. Mid-Log Suspension: Ideally, the mid-log suspension should be prepared 3-4 hours before class on Day 2. Alternatives that yield less than ideal results are: 1) growing bacteria to mid-log (3-4 hours of incubation) and then placing them in the refrigerator until the next day, or 2) letting the bacteria incubate on the countertop over night and then placing them in an incubator for a hour or two before use. If the students cannot be there after incubation, the teacher can perform the calcium chloride treatment and let it sit on ice overnight. Materials for Iowa teachers are supplied by The Office of Biotechnology, Iowa State University or teachers can order individual supplies from a scientific supply company. 6
PRE-LAB PREPARATIONS: Supplies are for each group of three students. DAY 1-Plasmid Digests 1. For each group aliquot: 5.5 µl of 0.20 µg/µl pamp in a tube marked pamp (store on ice) 5.5 µl of 0.20 µg /µl pkan in a tube marked pkan (store on ice) 20 µl of 2X restriction buffer in a tube labeled 2X RB - mix 20 µl of 10X restriction buffer with 80 µl of distilled water 6.0 µl of BamHI/HindIII enzymes in a tube labeled B/H - mix 3 µl of BamHI with 3 µl of HindIII in a 1.5 ml tube and store on ice 2. Adjust water bath to 37 o C. 3. Prepare a streak culture plate of MM294 E. coli. DAY 2- Mid-Log Bacterial Culture Materials: Procedure: MM294 bacteria 1-50 ml Conial tube Luria broth 1- sterile inoculating loop, bent paper clip or toothpick 37 o C water bath, shaking if possible, or incubator 2 sterile pipettes (glass or plastic) 1. Label a sterile 50 ml Conial tube with your name and date. 2. Do a sterile transfer of one pipette full of LB broth into the Conial tube. 3. Locate a well-formed colony from your plate of bacteria. Use a sterile inoculating loop, bent paper clip, or toothpick to scrape up the colony and transfer it to the LB broth in the tube. 4. Incubate the tube for 3-4 hours at 37 o C. Incubate 3 hours in a shaking water bath or 4 hours in the incubator with occasional shaking by hand. For best results, shake the tube a couple of times an hour when possible. 7
DAY 3- Ligation For each group of three students aliquot: 5 µl of 10X ligation buffer/atp labeled 10X LB/ATP 2 µl T4 DNA ligase labeled T4 Lig 15 µl distilled water labeled DW 1-1.5 ml tube 1-20 µl micropipettor and tips gloves (optional) permanent marker 65 o C water bath test tube racks 1-1.5 ml tube with 2 drops of sterile calcium chloride (on ice) labeled CaCl 2 Starter plate of bacteria (prepared on Day 1 of lab) 8
STUDENT LAB DAY 1 PLASMID DIGESTS Materials: 1 tube of pamp 1 tube of pkan 1 tube of BamHI/HindIII labeled B/H 1 tube of 2X restriction buffer labeled 2X RB 2-20 µl micropipettor and tips gloves (optional) permanent marker 37 o C water bath test tube racks 1 LB agar plate MM294 E. coli bacteria Procedure: 1. Obtain labeled 1.5 ml reaction tube containing pamp and pkan 2. Use the matrix below as a checklist while adding reagents to each tube. Use a fresh pipette tip for each reagent. Add all reagents directly to the solution. NOTE: The buffer is always added before the enzymes. BamHI/ Tube 2X Buffer HindIII ------------------------------------------------------------------------------------------------------------- pamp (5.5 µl) 7.5 µl 2 µl pkan (5.5 µl) 7.5 µl 2 µl 3. Place the reaction tubes in a 37 o C water bath and incubate for 30 minutes or longer. 4. After incubation, freeze the reaction at -20 o C until you are ready to proceed to Ligation (Day 3). 9
STUDENT LAB DAY 2 COMPETENT CELL PROCEDURE NOTE: The mid-log culture should be prepared 3-4 hours before class. The following supplies can be shared by groups of three students. Materials: Mid-log culture 1 ml Calcium Chloride CaCl 2 1-sterile 1.5 ml tube 3-sterile pipettes-glass or plastic Microcentrifuge (MicroV) or clinical centrifuge 1. From the Mid-Log suspension add 1 ml of culture to a 1.5 ml tube and microcentrifuge (MicroV microcentrifuge) at 10,000 RPM for 2 minutes. For centrifuges that spin at a slower RPM, you can adjust the time you spin the tube. The slower the RPM, the longer you should spin the tubes. Example: 10 minutes at 4000 RPM. 2. Sterilely drain off the LB broth, leaving a visible pellet of bacteria in the tube. 3. Use a sterile plastic pipette to transfer 0.5 ml (10 drops) of calcium chloride to the tube. 4. Resuspend the pelleted cell by vigorously vortexing the tube with your fingers. 5. Place on ice for 20 minutes. 6. Microcentrifuge the tube at 10,000 RPM for 2 minutes. 7. Sterilely drain off the calcium chloride, being careful not to disturb the cell pellet. 8. Sterilely add 2 drops of fresh calcium chloride and finger vortex to resuspend. 9. Place cells on ice in the refrigerator (approx. 0 o C) and store until you are ready to use them. DO NOT FREEZE. Holding the cell at 0 o C for 24 hours increases the cell competency. Cells can be held on wet ice for several days while you make the recombinant constructs. 10
STUDENT LAB DAY 3 LIGATION Materials: Procedure: 1 tube digested pamp 1 tube digested pkan 1 tube 10X ligation buffer/atp labeled 10X LB/ATP 1 tube T4 DNA ligase labeled T4 Lig 1 tube distilled water 1 tube 1.5 ml tube 2-20 µl micropipettor and tips gloves (optional) permanent marker 65 o C water bath test tube racks 1. Place the tubes marked pamp and pkan in a 65 o C water bath for 10 minutes. 2. Label a clean 1.5 ml tube Lig for ligation reaction. 3. Use the matrix below as a checklist while adding reagents to the ligation reaction. Make sure to add the reactants in the order listed from left to right. Add all reagents directly into the solution. Use a fresh pipette tip for each reagent. Digested Digested 10X Ligation Tube pamp pkan Water Buffer/ATP DNA Ligase ------------------------------------------------------------------------------------------------------------------ Lig 3 µl 3 µl 11 µl 4 µl 2 µl 4. Incubate the reaction at room temperature for 24 hours. IMPORTANT: Make sure you do not throw away the tube marked Lig when you dispose of your other reagent tubes. 5. If necessary, the ligation can be stored at -20 o C after incubation. Thaw the reaction before proceeding to transformation. For the best results, the transformation should take place immediately after incubation (the next day). Timing the events in the lab is very important. 6. Proceed to the Recombinant Transformation (Day 4) of the lab when your teacher instructs you to do so. 11
STUDENT LAB DAY 4 RECOMBINANT TRANSFORMATION Materials: 1 LB No Amp/Kan agar plate 1 LB Amp/Kan agar plate 1 LB Amp agar plate 1 LB Kan agar plate LB broth 37 o C incubator Spreader- bent paper clips 5 sterile transfer pipettes- Pasteur or polypropylene 2-20 µl micropipettors and tips 1.5 ml tube of competent cells Ligation solution Lig 1. Finger flick tube to resuspend cells. 2. Open the tube labeled CaCl 2 and add 20 µl of the ligation Lig directly to solution using a micropipettor and sterile tip. Close the tube. CaCl 2 3. Place the tube on ice for 15 minutes. CaCl 2 ICE 12
4. Remove the tube from the ice and immediately hold it in a 42 o C water bath for 90 seconds. Place the tube directly back on ice for 1 minute. (The marked temperature change causes the cells to readily absorb the plasmid DNA). 5. Use a sterile pipette to add 10 drops of sterile LB nutrient broth to the tube. Close the tube. Mix by tipping the tube and inverting it gently. (The bacteria are provided nutrients to help them recover from the calcium chloride and heat shock treatments). 6. If time permits, incubate the mixture for 3-4 hours at 37 o C. The teacher can remove the tube from the incubator and hold at room temperature overnight. The alternative is to continue to incubate the mixture overnight. STUDENT LAB DAY 5 RECOMBINANT TRANSFORMATION PLATING 1. Label the underside of the four petri dishes with your name and date. 2. Use a fresh sterile pipette and draw all the cell suspension from the CaCl 2 tube, into the pipette. Place 3 drops of cell suspension onto the center of a petri dish labeled LB Amp/Kan, 3 drops to the center of a dish labeled LB Amp, 3 drops of cell suspension to the center of a dish labeled LB Kan, and 3 drops of cell suspension to the center of a dish labeled LB. Use a fresh sterile paper clip to spread the liquid evenly across the surface of each plate. Do not touch the part of the paper clip that comes in contact with the agar. CaCl 2 + Lig + LB CaCl 2 + Lig + LB LB Amp/Kan LB Amp LB Kan LB 13
3. Incubate the plates agar down for 2 hours and then invert (agar up) until the next day at 37 o C. DAY 6 RECOMBINANT TRANSFORMATION RESULTS LB LB Amp LB Kan LB Amp/Kan 1. Observe and record your results above according to your teacher s direction. 2. The plates can then be sealed with parafilm and stored in the refrigerator for later observations. 14