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LABORATORY 3: Site-Directed Mutagenesis of Blue FluorescentProteinPart I (Monday Afternoon)

We are grateful to the Dolan DNA Learning Center at Cold Spring Harbor Laboratory andto 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 site2. 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 Streak a plate of mutagenesis of pBFPuv strain MM294

INTRODUCTION: Studies on the effects of mutations on organisms initially focused on thegeneration of random mutations in chromosomal DNA (such as those induced by X-rays andchemicals). Although these methods of random mutagenesis provided a valuable tool for classicalgenetic studies, the usefulness of the mutations was limited because it was not possible to target aspecific gene or genetic element. The random mutagenesis of an entire genome also requiredscreening 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 tothe genome. This method, known as site directed mutagenesis, earned its inventor Michael Smiththe 1993 Nobel Prize for Chemistry. This method, which employs plasmid vectors to introduce themodified DNA, became a driving force for newer technologies, which allowed precise changes indiscrete, manageable segments of the genome with relatively little effort.

The specific method we will use is a type of mutagenesis termed oligonucleotide-directedmutagenesis because a short sequence of bases, an oligonucleotide, encoding the desiredmutation(s) is annealed to one strand of the target DNA and also serves as a primer for initiation ofDNA synthesis. The primers used for this method must meet certain requirements. The twoprimers must both contain the desired mutation and must anneal to the same sequence on bothstrands of the plasmid. In addition, the desired mutation must be in the middle of the primersequence 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 theplasmid DNA is denatured, producing two complementary single-stranded rings of DNA to whichthe respective primers anneal. The Taq DNA polymerase then extends the primer sequence until acomplete 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 nickedcircles, but there will also be some unmutated plasmid present. This mixture is then transformedinto competent E. coli cells, which have been treated with calcium ions to allow them to take upthe plasmid DNA. Once the DNA has been taken up by the cells, the nicks in the plasmids arerepaired intracellularly, and the plasmids are able to replicate and express the mutated protein.

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Once the transformation has been completed, the bacteria are plated on selective plates to selectfor the E. coli cells that have taken up a plasmid, usually by requiring that these cells express anampicillin resistance gene that was inserted into the plasmid. After the cells have grown intocolonies on the plates, the colonies are screened to determine which colonies actually contain themutated gene. Mutagenic oligonucleotides incorporate at least one base change but can bedesigned 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 UVillumination. In the early 1960s, Shimomura and Johnson at Princeton University studied thesource of bioluminescence in jellyfish. One of the compounds they discovered was GFP, whichthey isolated from the bioluminescent jellyfish Aequoria Victoria (Fig. 2). Subsequent studies

showed that GFP can be expressed without any additional enyzmes or cofactors from theorganism. Consequently, if the coding sequence for GFP is incorporated into a vector, it is

Figure 2: A. Victoria jellyfish

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possible to express GFP in cells from various species. This has led to the use of GFP as a monitorof gene expression and protein localization. GFP is a protein of 238 amino acids, which forms acylindrical structure called a -can (Fig. 3). The overall structure is shown on the left in theFigure below where the protein chain forms the cylindrical can (blue) with a portion of the strandrunning through the middle of the can (green). The light absorbing and emitting portion of themolecule, termed the chromophore or fluorophore, is on the middle of this strand (white), thisstructure forms an important function because the fluorophore is protected by the can fromcollisions with water molecules that would otherwise deactivate the excited molecule before itcould emit light (quenching). On the right is a detailed image of the fluorophore itself that showsthe 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 tomake 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 themolecule can change other characteristics including its solubility and absorption spectrum. In thisexperiment, you will make specific changes to the BFP coding sequence to change the color of thefluorescence emitted by the mutant protein. You will use a vector derived from pUC19, a popularvector that was constructed in the 1980s from a naturally occurring E. coli plasmid usingrecombinant DNA techniques. pUC19 has several features important in cloning including areplication origin that enables it to replicate independently of the host chromosome and a genefor ampicillin resistance (Ampr) that is used to select for the presence of pUC19 in a cell. Thesequence for BFP has been inserted into this vector. You will use a commercially availableplasmid, a map of one of which, pBFPuv, is shown below (Fig. 4). Table 1 on the next page showsthe color change that results from the various changes in the amino acid at position 66 (primermismatch shown in red). A shorthand notation for the mutation is often used, the BFPuv constructis referred to as H66Y.

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PlasmidOrig. 66Sequence

New 66Sequence

Newcolor Primers

pBFP2(blue)

CATHistidine

TATTyrosine

Green5' GTC ACT ACT TTG ACC TAT GGT GTT CAA TGC5' GCA TTG AAC ACC ATA GGT CAA AGT AGT GAC

Figure 4: Restriction Map and Multiple Cloning Site (MCS) of pBFPuv Vector (Clontech)

pBFPuv 3.3 kb

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II. EXPERIMENTAL PROCEDURES: There are threebasic steps in the process that you will start today. First, thevector DNA is mutated using site directed mutagenesis tochange a specific nucleotide. Second, E. coli MM294 cells thatyou have treated with calcium ions to make them competent aretransformed with the mutation mix and plated onto specialselective plates to select for the E. coli cells that have taken upthe plasmid. Third, after the cells grow into bacterial colonieson these plates, the colonies are examined to determine whichcolony or colonies has the clone containing the mutagenizedDNA sequence of interest.

A. Setting up a PCR Reaction to Mutate the BFP-Containing Plasmid pBFPuv: Each lab. pair should worktogether 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 0.2ml thin-walled PCR reaction tube.

3. Add 25 l of master mix which is at the front bench to thisPCR bead tube.

> The master mix includes primers,water and the pBFP plasmidDNA.

4. Carefully flick the mixture you have just made to ensure goodmixing.

5. Transfer this 28 l mixture to the 0.2 ml thin walled PCRtube (blue).

6. Use a marker to write your initials on the side of this bluePCR tube and put the tube on ice in a PCR rack.

7. Pulse spin using a black adapter then take this reaction tube toa member of the lab staff who will load all your tubes into thethermal cycler for 25 cycles of the following conditions:

Denaturation 94C 5 min

25 cycles: Denaturing 94C 30 sec

Primer Annealing 58C 30 sec

Synthesis 72C 4 min

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Final synthesis 72C 7 min

Holding 4C indefinitely

After the completion of these reactions, your tube will be storeduntil tomorrow afternoon, when you will transform your PCRproducts into E. coli.

B. Basic Microbial Techniques: The aseptic techniques you willperform next are fundamental procedures in molecular biologyand are often used when working with bacteria and othermicroorganisms. Because you will again use sterile technique,make sure the surface of your bench is clean and organize yourmaterials before beginning. Each person should perform thefollowing exercises.

> Be sure to position the Bunsenburner so that you do not have toreach over it.

1. Why Use Sterile Technique? To demonstrate to yourself theimportance of working carefully and using good sterile techniquein your experiments, each person should obtain one LB plate (onered stripe on the side) from the bench at the front of the lab.

> For good technique, do not putsterile items down on the labbench--it is not sterile.

a. Label the bottom of the plate with your initials using a blackmarker.

b. Remove the lid of the plate and wipe the palm of one handover the entire surface of the agar.

c. Replace the lid, tape this plate closed, and place it in a plasticbin on the front table.

> Your plate will be incubatedovernight. You will assess thesterility of your hand tomorrow.

2. Isolation of Individual Bacterial Colonies: Streaking is atechnique used to isolate individual bacterial colonies on solidmedium. You will work with the Escherichia coli strain MM294that is described in DNA Science, 2nd Ed. You and your partnerwill share the stock plate of MM294 that is at your bench.

> Streaking is described below andin Laboratory 2 (Part A), D N AScience 2nd Ed.

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 yourname or initials and MM294.

c. Turn on the gas and light your Bunsen burner. > If it is burning correctly, the flameshould have a blue cone in thecenter.

d. Holding your inoculating loop like a pencil, heat the loop atthe top of the inner blue cone until the loop glows bright red.

> The top of the blue cone is thehottest part of the flame.

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e. Remove the lid from the stock plate of MM294, holding thelid face down just above the plate instead of putting the liddown on your bench.

> This helps prevent air-bornecontaminants from falling onto theagar or in the lid.

f. Cool the loop by gently touching it to the surface of the agarnear the side of the plate.

> What is the purpose of cooling theloop?

g. Use a sweeping motion of your loop to pick up part of abacterial 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 streakwith 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 andcontinue streaking in a tight zigzag pattern to the bottom ofthe plate.

> Do not lift the loop or crossanother part of the original streakas shown below:

l. Using this streaking technique will decrease the number ofbacteria on the loop as you streak, so individual colonies willgrow near the bottom of the streak after incubation.

m. Tape your plate to that of your partner and place themupside down in the 37o C incubator for overnightincubation.

> Why are the plates incubatedupside down?

After you have performed this streaking technique a few timesand feel comfortable with it, you can conserve plates and mediaby streaking 8 10 strains onto a single plate.

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LABORATORY 3: Site-Directed Mutagenesis of Blue FluorescentProteinPart II (Tuesday Afternoon)

Objectives of Laboratory 3, Part II:1. Prepare competent MM294 cells2. Transform these competent cells with your mutated DNA3. Plate cells to select for transformants and identify mutant plasmids

Flow Chart of Laboratory 3, Part II: Prepare competent Transform these Plate transformed cells MM294 cells competent cells to selective plates

II. EXPERIMENTAL PROCEDURES: Today youwill mix your PCR products (mutation mix) with competentMM294 E. coli cells.

A. Rapid Preparation of Competent MM294 Cells: Thisrapid, quick and dirty method uses colonies of MM294 grownon a freshly streaked LB plate, which you prepared yesterdayand incubated overnight at 37o C.

1. Each lab pair should prepare cells from the fresh plate ofMM294 they prepared yesterday as described next.

2. Obtain an Isotherm, some ice, and a clear 1.5 ml microtubecontaining 200 l 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 theplate.

5. Use your loop to transfer 2 large loopfuls of MM294 (thismay consist of several colonies) from your plate to the iced 50mM CaCl2.

> The calcium ions make the cellwall of E. coli permeable so thecells can take up DNA fromsolution.

6. Disrupt these colonies by repeatedly pipetting the solution upand 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.

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9. When the cells are thoroughly and uniformly resuspended inthe CaCl2, put this tube on ice again.

> The E. coli cells become fragilewhen they are treated withcalcium, so keep them on ice andtreat them gently at all times.

B. Preparation of Your Transformation Tube:

1. Take your ice bucket to the front of the lab and obtain yourmutation mix (blue microtube) and your BFP/GFP PCR producttube.

2. Add 20 l of your PCR product to the tube containing thecompetent 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 42C waterbath for 90 seconds to heat shock the cells.

> This heat shock is required forcells to take up DNA fromsolution.

6. Remove the tube from the water bath to a test tube rack.

7. Add 0.8 ml of sterile LB growth medium to yourtransformation tube using sterile technique.

> The LB is in 15 ml orange-cappedtubes.

12. Spin this tube for 10 min at 2000 rpm. > It is important not to exceed thisspeed.

C. Plating Your Transformation Mix to Selective Plates:While you are centrifuging your transformation tube, obtain oneplate that contains selective growth medium (LB agar +ampicillin) from the front bench.

> These plates have one black stripeon their sides.

1. Label this plate with BFP/GFP and your initials.

2. When your transformation tube has finished spinning removeabout 800 l of supernatant and resuspend the bacterial pellet inthe remaining liquid (~200 l).

> Use your fingers or a pipette togently resuspend the pellet. D onot vortex!

3. Pour the contents of your BFP/GFP tube onto the surface ofthe plate labeled BFP/GFP.

4. Light the Bunsen burner at your bench with the strikerprovided and remove the top from the jar of alcohol.

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5. Dip the glass rod into the alcohol and immediately put the rodinto the flame of the burner briefly to ignite the alcohol.

6. Quickly remove the rod from the flame as soon as the alcoholignites.

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 tothe agar near the side of the dish.

10. Then, move the rod in a circular motion across the surfaceof the agar to spread the liquid evenly over the entire surface.

11. Continue moving the glass rod in a circular motion until allthe liquid is absorbed into the agar, and the surface of the agarappears dry.

12. Replace the top of the Petri dish.

13. Put your plate upside down into the 37C incubator to growovernight.

> During this incubation period,each E. coli cell will grow into avisible colony or clone of cells.

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LABORATORY 3: Site-Directed Mutagenesis of Blue FluorescentProteinPart III (Wednesday Morning)

Objectives of Laboratory 3, Part III:1. Observe your transformation plate and count the colonies present2. Identify and count mutant plasmids

Flow Chart of Laboratory 3, Part III: Count the colonies present on Record the numbers of your transformation plate colonies of different colors

I. EXPERIMENTAL PROCEDURES:

1. Count the colonies on your plate. > Use a black marker to mark thecolonies on the back of the plateas you count

2. Observe the plate under UV light and record yourobservations.

3. Record the number of colonies of each color that are presenton each plate.