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LABORATORY 3: Site-Directed Mutagenesis of Blue Fluorescent Protein Part I (Monday Afternoon) We are grateful to the Dolan DNA Learning Center at Cold Spring Harbor Laboratory and to Dr. Jennifer Aizenmann for making this protocol available to us. Objectives of Laboratory 3, Part I: 1. Prepare a PCR reaction to mutate a BFP-carrying plasmid at a specific site 2. Streak E. coli strain MM294 to a fresh plate to use to prepare competent cells tomorrow Flow Chart of Laboratory 3, Part I: Prepare a PCR reaction for mutagenesis of pBFPuv Streak a plate of strain MM294 INTRODUCTION: Studies on the effects of mutations on organisms initially focused on the generation of random mutations in chromosomal DNA (such as those induced by X-rays and chemicals). Although these methods of random mutagenesis provided a valuable tool for classical genetic studies, the usefulness of the mutations was limited because it was not possible to target a specific gene or genetic element. The random mutagenesis of an entire genome also required screening or selection from massive numbers of mutants to obtain the desired mutation. However, with the advent of recombinant DNA techniques, it became possible to make specific changes to the genome. This method, known as site directed mutagenesis, earned its inventor Michael Smith the 1993 Nobel Prize for Chemistry. This method, which employs plasmid vectors to introduce the modified DNA, became a driving force for newer technologies, which allowed precise changes in discrete, manageable segments of the genome with relatively little effort. The specific method we will use is a type of mutagenesis termed oligonucleotide-directed mutagenesis because a short sequence of bases, an oligonucleotide, encoding the desired mutation(s) is annealed to one strand of the target DNA and also serves as a primer for initiation of DNA synthesis. The primers used for this method must meet certain requirements. The two primers must both contain the desired mutation and must anneal to the same sequence on both strands of the plasmid. In addition, the desired mutation must be in the middle of the primer sequence and be flanked by about 12 bases on either side. The primers should also be at least 40% GC and should terminate in at least one C or G. The process used for this mutagenesis is illustrated in Figure 1 on the next page. First the plasmid DNA is denatured, producing two complementary single-stranded rings of DNA to which the respective primers anneal. The Taq DNA polymerase then extends the primer sequence until a complete circle of DNA is synthesized. This circle however, is not sealed because a nick remains. If this process is repeated enough times, all the primer molecules can be converted to nicked circles, but there will also be some unmutated plasmid present. This mixture is then transformed into competent E. coli cells, which have been treated with calcium ions to allow them to take up the plasmid DNA. Once the DNA has been taken up by the cells, the nicks in the plasmids are repaired intracellularly, and the plasmids are able to replicate and express the mutated protein. 35 Once the transformation has been completed, the bacteria are plated on selective plates to select for the E. coli cells that have taken up a plasmid, usually by requiring that these cells express an ampicillin resistance gene that was inserted into the plasmid. After the cells have grown into colonies on the plates, the colonies are screened to determine which colonies actually contain the mutated gene. Mutagenic oligonucleotides incorporate at least one base change but can be designed to generate multiple substitutions, insertions or deletions. Figure 1: Schematic of the Site Directed Mutagenesis process, (Royal Swedish Academy of Sciences) The subject of the mutagenesis in the experiment you will perform is Blue Fluorescent Protein (BFP), a variant of Green Fluorescent Protein, which fluoresces bright green under UV illumination. In the early 1960s, Shimomura and Johnson at Princeton University studied the source of bioluminescence in jellyfish. One of the compounds they discovered was GFP, which they isolated from the bioluminescent jellyfish Aequoria Victoria (Fig. 2). Subsequent studies Figure 2: A. Victoria jellyfish showed that GFP can be expressed without any additional enyzmes or cofactors from the organism. Consequently, if the coding sequence for GFP is incorporated into a vector, it is 36 possible to express GFP in cells from various species. This has led to the use of GFP as a monitor of gene expression and protein localization. GFP is a protein of 238 amino acids, which forms a cylindrical structure called a ‘β-can’ (Fig. 3). The overall structure is shown on the left in the Figure below where the protein chain forms the cylindrical can (blue) with a portion of the strand running through the middle of the can (green). The light absorbing and emitting portion of the molecule, termed the chromophore or fluorophore, is on the middle of this strand (white), this structure forms an important function because the fluorophore is protected by the can from collisions with water molecules that would otherwise deactivate the excited molecule before it could emit light (quenching). On the right is a detailed image of the fluorophore itself that shows the three amino acids, numbers 65, 66 and 67, that are involved in the generation of fluorescence. In the wild type GFP these amino acids are serine (or threonine), tyrosine and glycine. Figure 3: Structure of GFP (Protein Data Bank) Recently, attempts have been made to alter the spectral characteristics of the protein in order to make it useful for a wider variety of applications. By changing the amino acids of the fluorophore, it is possible to change the color of the fluorescence. Amino acid changes made elsewhere in the molecule can change other characteristics including its solubility and absorption spectrum. In this experiment, you will make specific changes to the BFP coding sequence to change the color of the fluorescence emitted by the mutant protein. You will use a vector derived from pUC19, a popular vector that was constructed in the 1980’s from a naturally occurring E. coli plasmid using recombinant DNA techniques. pUC19 has several features important in cloning including a replication origin that enables it to replicate independently of the host chromosome and a gene for ampicillin resistance (Ampr) that is used to select for the presence of pUC19 in a cell. The sequence for BFP has been inserted into this vector. You will use a commercially available plasmid, a map of one of which, pBFPuv, is shown below (Fig. 4). Table 1 on the next page shows the color change that results from the various changes in the amino acid at position 66 (primer mismatch shown in red). A shorthand notation for the mutation is often used, the BFPuv construct is referred to as H66Y. 37 Plasmid Orig. 66 Sequence New 66 Sequence New color pBFP2 (blue) CAT Histidine TAT Tyrosine Green 5' GCA TTG AAC ACC ATA GGT CAA AGT AGT GAC Primers 5' GTC ACT ACT TTG ACC TAT GGT GTT CAA TGC pBFPuv 3.3 kb Figure 4: Restriction Map and Multiple Cloning Site (MCS) of pBFPuv Vector (Clontech) 38 II. EXPERIMENTAL PROCEDURES: There are three basic steps in the process that you will start today. First, the vector DNA is mutated using site directed mutagenesis to change a specific nucleotide. Second, E. coli MM294 cells that you have treated with calcium ions to make them competent are transformed with the mutation mix and plated onto special selective plates to select for the E. coli cells that have taken up the plasmid. Third, after the cells grow into bacterial colonies on these plates, the colonies are examined to determine which colony or colonies has the clone containing the mutagenized DNA sequence of interest. A. Setting up a PCR Reaction to Mutate the BFPContaining Plasmid pBFPuv: Each lab. pair should work together to set up one PCR reaction. 1. Obtain an Isotherm and some ice. 2. From the front bench, collect a PCR bead tube and a blue 0.2 ml thin-walled PCR reaction tube. 3. Add 25 µl of master mix which is at the front bench to this > The master mix includes primers, water and the pBFP p l a s m i d PCR bead tube. DNA. 4. Carefully flick the mixture you have just made to ensure good mixing. 5. Transfer this 28 µl mixture to the 0.2 ml thin walled PCR tube (blue). 6. Use a marker to write your initials on the side of this blue PCR tube and put the tube on ice in a PCR rack. 7. Pulse spin using a black adapter then take this reaction tube to a member of the lab staff who will load all your tubes into the thermal cycler for 25 cycles of the following conditions: Denaturation 25 cycles: 94ºC 5 min Denaturing 94ºC 30 sec Primer Annealing 58ºC 30 sec Synthesis 72ºC 4 min 39 Final synthesis Holding 72ºC 7 min 4ºC indefinitely After the completion of these reactions, your tube will be stored until tomorrow afternoon, when you will transform your PCR products into E. coli. B. Basic Microbial Techniques: The aseptic techniques you will > Be sure to position the Bunsen burner so that you do not have to perform next are fundamental procedures in molecular biology reach over it. and are often used when working with bacteria and other microorganisms. Because you will again use sterile technique, make sure the surface of your bench is clean and organize your materials before beginning. Each person should perform the following exercises. 1. Why Use Sterile Technique? To demonstrate to yourself the > For good technique, do not put sterile items down on the lab importance of working carefully and using good sterile technique bench--it is not sterile. in your experiments, each person should obtain one LB plate (one red stripe on the side) from the bench at the front of the lab. a. Label the bottom of the plate with your initials using a black marker. b. Remove the lid of the plate and wipe the palm of one hand over the entire surface of the agar. c. Replace the lid, tape this plate closed, and place it in a plastic > Your plate will be incubated overnight. You will assess the bin on the front table. sterility of your hand tomorrow. 2. Isolation of Individual Bacterial Colonies: Streaking is a > Streaking is described below and in Laboratory 2 (Part A), D N A technique used to isolate individual bacterial colonies on solid Science 2nd Ed. medium. You will work with the Escherichia coli strain MM294 that is described in DNA Science, 2nd Ed. You and your partner will share the stock plate of MM294 that is at your bench. a. Each person should obtain an LB plate (one red stripe on side) from the front of the lab if it is not at your bench: b. On the bottom of this plate, use a black marker to write your name or initials and MM294. c. Turn on the gas and light your Bunsen burner. > If it is burning correctly, the flame should have a blue cone in the center. d. Holding your inoculating loop like a pencil, heat the loop at > The top of the blue cone is the hottest part of the flame. the top of the inner blue cone until the loop glows bright red. 40 e. Remove the lid from the stock plate of MM294, holding the > This helps prevent air-borne contaminants from falling onto the lid face down just above the plate instead of putting the lid agar or in the lid. down on your bench. f. Cool the loop by gently touching it to the surface of the agar > What is the purpose of cooling the loop? near the side of the plate. g. Use a sweeping motion of your loop to pick up part of a bacterial colony from the MM294 stock plate. h. Replace the lid of this stock plate. i. Remove the lid of your LB plate and make a single streak with your loop at the top of the plate as shown to the right. j. Flame your loop again and cool it in the agar in this plate. k. Pass your loop through your first streak only once and > Do not lift the loop or cross another part of the original streak continue streaking in a tight zigzag pattern to the bottom of as shown below: the plate. l. Using this streaking technique will decrease the number of bacteria on the loop as you streak, so individual colonies will grow near the bottom of the streak after incubation. m. Tape your plate to that of your partner and place them > Why are the plates incubated upside down? upside down in the 37o C incubator for overnight incubation. After you have performed this streaking technique a few times and feel comfortable with it, you can conserve plates and media by streaking 8 – 10 strains onto a single plate. 41 LABORATORY 3: Site-Directed Mutagenesis of Blue Fluorescent Protein Part II (Tuesday Afternoon) Objectives of Laboratory 3, Part II: 1. Prepare competent MM294 cells 2. Transform these competent cells with your mutated DNA 3. Plate cells to select for transformants and identify mutant plasmids Flow Chart of Laboratory 3, Part II: Prepare competent MM294 cells Transform these competent cells Plate transformed cells to selective plates II. EXPERIMENTAL PROCEDURES: Today you will mix your PCR products (mutation mix) with competent MM294 E. coli cells. A. Rapid Preparation of Competent MM294 Cells: This rapid, “quick and dirty” method uses colonies of MM294 grown on a freshly streaked LB plate, which you prepared yesterday and incubated overnight at 37o C. 1. Each lab pair should prepare cells from the fresh plate of MM294 they prepared yesterday as described next. 2. Obtain an Isotherm, some ice, and a clear 1.5 ml microtube containing 200 µl sterile, iced 50 mM CaCl2. 3. Flame your loop in the burner flame until it glows red. 4. Cool your loop slightly by touching it to a clear area of the plate. 5. Use your loop to transfer 2 large loopfuls of MM294 (this > The calcium ions make the cell wall of E. coli permeable so the may consist of several colonies) from your plate to the iced 50 cells can take up DNA from mM CaCl2. solution. 6. Disrupt these colonies by repeatedly pipetting the solution up and down with your P200, which may take a couple of minutes. 7. Transfer two more loopfuls of cells to the tube of iced CaCl2. 8. Use your P200 again to break up these colonies by pipetting. 42 9. When the cells are thoroughly and uniformly resuspended in > The E. coli cells become fragile when they are treated with the CaCl2, put this tube on ice again. calcium, so keep them on ice and treat them gently at all times. B. Preparation of Your Transformation Tube: 1. Take your ice bucket to the front of the lab and obtain your mutation mix (blue microtube) and your BFP/GFP PCR product tube. 2. Add 20 µl of your PCR product to the tube containing the competent cells. 3. Then, flick this tube gently with your fingers. 4. Let this tube sit on ice for 30 minutes. 5. After the 30 min on ice ends, put the tube into the 42ºC water > This heat shock is required for cells to take up DNA from bath for 90 seconds to heat shock the cells. solution. 6. Remove the tube from the water bath to a test tube rack. 7. Add 0.8 ml of sterile LB growth medium to your > The LB is in 15 ml orange-capped tubes. transformation tube using sterile technique. 12. Spin this tube for 10 min at 2000 rpm. > It is important not to exceed this speed. C. Plating Your Transformation Mix to Selective Plates: > These plates have one black stripe on their sides. While you are centrifuging your transformation tube, obtain one plate that contains selective growth medium (LB agar + ampicillin) from the front bench. 1. Label this plate with “BFP/GFP” and your initials. 2. When your transformation tube has finished spinning remove > Use your fingers or a pipette to gently resuspend the pellet. D o about 800 µl of supernatant and resuspend the bacterial pellet in not vortex! the remaining liquid (~200 µl). 3. Pour the contents of your “BFP/GFP” tube onto the surface of the plate labeled “BFP/GFP”. 4. Light the Bunsen burner at your bench with the striker provided and remove the top from the jar of alcohol. 43 5. Dip the glass rod into the alcohol and immediately put the rod into the flame of the burner briefly to ignite the alcohol. 6. Quickly remove the rod from the flame as soon as the alcohol ignites. 7. A couple seconds after you remove the rod from the flame, the flame will go out. 8. This process of flaming sterilizes the glass rod. 9. Take the top off the plate and cool the rod by touching it to the agar near the side of the dish. 10. Then, move the rod in a circular motion across the surface of the agar to spread the liquid evenly over the entire surface. 11. Continue moving the glass rod in a circular motion until all the liquid is absorbed into the agar, and the surface of the agar appears dry. 12. Replace the top of the Petri dish. 13. Put your plate upside down into the 37ºC incubator to grow overnight. 44 > During this incubation period, each E. coli cell will grow into a visible colony or clone of cells. LABORATORY 3: Site-Directed Mutagenesis of Blue Fluorescent Protein Part III (Wednesday Morning) Objectives of Laboratory 3, Part III: 1. Observe your transformation plate and count the colonies present 2. Identify and count mutant plasmids Flow Chart of Laboratory 3, Part III: Count the colonies present on your transformation plate Record the numbers of colonies of different colors I. EXPERIMENTAL PROCEDURES: 1. Count the colonies on your plate. > 2. Observe the plate under UV light and record your observations. 3. Record the number of colonies of each color that are present on each plate. 45 Use a black marker to mark the colonies on the back of the plate as you count