Download LABORATORY 3: Site-Directed Mutagenesis of Blue

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.
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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
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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.
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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)
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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
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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.
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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.
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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.
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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.
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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.
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>
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.
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Use a black marker to mark the
colonies on the back of the plate
as you count