BIOL3422 Experiment 4: Bacterial Transformation PDF

Experiment 4: Bacterial Transformation After this lab, students should be able to: Understand the concept of horizontal gene transfer. Understand and perform a bacterial transformation experiment. Explain the use of positive and negative controls in an experiment. Calcul...

Experiment 4: Bacterial Transformation After this lab, students should be able to: Understand the concept of horizontal gene transfer. Understand and perform a bacterial transformation experiment. Explain the use of positive and negative controls in an experiment. Calculate transformation e>iciency. Background In this lab we will begin studying methods of bacterial horizontal gene transfer. Horizontal gene transfer is the term used to describe any method where bacteria share genetic information with each other. This contrasts with vertical gene transfer, where genetic information is passed from parent to o>spring. The three main methods of horizontal gene transfer between bacteria are transformation, conjugation, and transduction. Horizontal gene transfer is one of the main causes of the spread of antibiotic resistances among bacteria, but it is also utilized by microbiologists for genetic manipulation of bacterial cultures. Bacterial transformation is a process by which a recipient bacterial cell takes up DNA from its environment. Today, we will be using DNA transformation to introduce an antibiotic resistance plasmid into chemically competent E. coli recipient cells. Competence Competence is the ability of a bacteria or other microorganism to take up DNA from its environment. Some bacteria are naturally competent due to their genetics, but E. coli is not among these species. For E. coli, competence can be artificially induced through a process called chemically competent transformation. In this process, plasmid DNA is mixed with cold cells that have been treated with calcium chloride (CaCl2). CaCl2 alters the cell membrane to make it more permeable to DNA, in addition to counteracting the negative charge of DNA molecules. Cells are then exposed to a high temperature for a short period of time, called a heat shock, which briefly disrupts the cell membrane and allows the plasmid DNA to enter. Cells are then put back on ice, provided nutrient broth, and left to recover. During this recovery period, bacteria that have taken up the plasmid will start to replicate it and express the genes within – in our case, resistance for the antibiotic ampicillin. Plasmids A plasmid is an extrachromosomal circular DNA element that can replicate independently of the bacterial host chromosome (recall that prokaryotic cells contain a single, looped chromosome). Plasmids are evolutionarily important because they allow for horizontal gene transfer between bacterial cells and may confer selective advantage under stressful conditions. For example, the plasmid we will be using today, pAMP, contains the gene bla that will confer resistance to the antibiotic ampicillin to a host that expresses the plasmid genes. In addition to their roles in horizontal gene transfer, plasmids are widely used in recombinant DNA technologies. Designing Controls In any scientific experiment, a good design includes experimental controls that indicate whether the technical procedures used are correctly working. It is not uncommon for something to go wrong in science, and having e>ective controls will help you determine whether the result you see is due to an issue with the procedure, reagents, or techniques used. Positive controls show how results would look if an experiment worked properly. If your experiment’s positive control does not produce the expected result, that is an indication there may be an issue with the protocol or a confounding variable you forgot to address. Having an e>ective positive control allows you to establish that the protocol is working as expected. Negative controls show what the result of an experiment would be if it did not work. If you get a positive result in your positive control AND a positive result in your negative control, that indicates there is some confounding variable you did not address. Having an e>ective negative control allows you to establish a baseline for success in your experiments. Pay attention to the positive and negative controls in this experiment, and all experiments going forward! Before starting an experiment, ask yourself what each control is testing for, and what you expect to see in each control. This will help you interpret your results down the road. Purpose To introduce by transformation a plasmid containing resistance to Ampicillin into a culture of E. coli Materials (for each pair of students): Tube of competent cells, prepared by your instructors. KEEP THIS ON ICE. Tube of pAMP (at a concentration of 1µg/mL). KEEP THIS ON ICE. 2 LB plates containing ampicillin (at a concentration of 100µg/mL) – these are labeled with an “A” 2 LB plates without any antibiotics 1 mL of LB broth Glass beads for spreading Ice bucket with ice Additionally, students will share a shaking incubator set to 37°C and a water bath set to 42°C. Protocol: Preparation of Competent Cells Prior to class, chemically competent E. coli cells were prepared using the following steps: 1. Dilute an overnight culture of E. coli 1:100 in fresh liquid LB and grow in a shaking incubator set to 37°C for 2-3 hours. The OD600 should read between 0.3 – 0.5 before proceeding. 2. Transfer the culture to sterile 50mL conical tubes and centrifuge at 5000RPM for 10 minutes at 4°C. Discard supernatant and place the pelleted cells on ice.  3. Resuspend the cells in 10mL of cold 0.1M CaCl2 solution. Centrifuge for 2500RPM for 5 minutes at 4°C. Discard supernatant and resuspend cells in an additional 10mL CaCl2. Sit on ice 30 minutes. 4. Centrifuge cells once more at 2500RPM for 5 minutes at 4°C. Discard supernatant and resuspend cells in 2mL cold CaCl2. These cells can be used for transformation right away, or frozen for future use. Transformation You will set up two tubes of cells for your transformation experiment. Tube A will contain competent cells and pAMP. Tube B will contain competent cells and water. Both tubes will go through the same transformation process, and you will plate both on LB and on LB+Amp. Develop a hypothesis: What do you expect to see on each of your plates, assuming the protocol goes correctly? LB + Tube A: _____________________________________________________________________ LB + Tube B: _____________________________________________________________________ LB/Amp + Tube A: _____________________________________________________________________ LB/Amp + Tube B: _____________________________________________________________________ Make sure to keep competent cells and plasmid on ice until told otherwise! Heating these cells, even with your hands, will lower the eFiciency of this experiment. 1. Label two 1.5mL Eppendorf tubes “A” and “B”. Chill on ice for 2 minutes. 2. In each tube, pipette 50µL of competent E. coli cells. 3. To tube A, add 10µL of pAMP DNA and carefully mix with your pipet. 4. To tube B, add 10µL of water and carefully mix with your pipet. 5. Incubate the tubes on ice for 30 minutes. 6. Heat shock the cells. Bring your ice bucket to the water bath. Move your cells from the ice bucket to the water bath for 90 seconds, then return them directly to the ice bucket. a. It is essential that you do this step quickly! The tubes should go directly from the ice bucket into the water bath, then directly back into the ice bucket. 7. Let the cells recover on ice for 2 minutes, then add 250µL of LB broth to each tube. Gently mix with a pipet. 8. Place the tubes in the 37°C shaker for at least an hour. a. During this time, you should look at the results of your environmental sample from last week, perform a gram stain, and look at it under the microscope. 9. Clearly your LB and LB+Amp plates. Retrieve your tubes from the shaker, and pipette 100µL onto each plate, using 8-10 glass beads to spread the bacteria evenly. Empty the glass beads into the burn-up bins. 10. Your plates will be incubated at 37°C for 24 – 48 hours, and we will look at their growth next lab. Data Analysis: Make a rough count of the number of colonies per plate and record the results in the table below. Do not count tiny “satellite” colonies that emerge on the edges of a larger colony. If there is lawn growth of cells, indicate that in your data table. Sample Plate Type # of colonies Expected Result 1. Calculate transformation e>iciency through the following steps. Transformation e>iciency is expressed as the number of colony forming units (CFUs) per µg of plasmid DNA. Remember that each CFU arose from a single cell and is a clonal population. Keep track of units throughout this process! a. Calculate the mass of plasmid DNA present in the transformation mixture (in µg). b. Calculate the concentration of plasmid DNA in the transformation mixture containing cells, LB, and the plasmid. c. Calculate the mass of plasmid DNA applied to the plate. d. Divide the number of colonies from this plate by mass of plasmid DNA added to the plate. This number is your transformation e>iciency, expressed in colonies per µg of plasmid DNA. 2. Explain the results of your control plates. Do they align with your hypotheses about what you expected? If there is any discrepancy between your hypotheses and results, suggest what this might mean about your experiment. Environmental Isolate Remember to look at your plates from last week to see how your environmental sample grew on MSA and EMB plates, and to answer the questions in experiment 3’s analysis. During the wait steps in today’s lab, perform a gram stain on your environmental samples, and re-streak onto LB agar once more. Next week, we will be starting a more intensive investigation of their properties, so make sure you have at least one pure culture between you and your lab partner!