NAME: ___________________________
Transformation (Pre)Lab Chart: Complete the columns 2-6 from the left as your pre-lab preparation. You
can use the completed table for your pre-lab, in-class quiz!
The Effect of ____________________ , ______________________ , _____________________ on ______________________
and ________________________ of E. coli that experienced a heat shock treatment to facilitate transformational competence
Plate
Name
Does the
Did the
Does the agar
agar
bacteria
medium
medium
receive the
contain
contain
plasmid?
ampicillin?
arabinose?
Is plate the
positive
control,
negative
control, or
experimental?
Prediction: Will
bacteria grow?
Will bacteria
glow green?
Sketch of how
plate will look
Results:
Draw actual #
& color of
bacteria
colonies (in
plain & UV
light)
E. coli
starter
-pGLO
LB
-pGLO
LB/amp
+pGLO
LB/amp
+pGLO
LB/amp/
ara
Additional Transformation Lab Data:
Plate
Color of
Color of
Size of
1
Size of
Transformation
Chi square value
colonies
under normal
light
colonies
under UV
light
largest
colony
(mm)
smallest
colony
(mm)
Efficiency
&
p-value
-pGLO
LB
-pGLO
LB/amp
+pGLO
LB/amp
+pGLO
LB/amp/ara
Class Data: Transformation Efficiency & Standard Deviation
Plate
Trial #
Transformation
Efficiency
Difference
from mean
squared
Sum of difference
squared / 7, AND
THEN find square
root = SD
Is group data more than two
SD from mean (yes or no).
If yes, provide an explanation
1
2
3
+pGLO
LB/amp
4
5
6
7
8
MEAN
1
2
3
+pGLO
LB/amp/ara
4
5
6
7
8
MEAN
AP Biology
PRE-LAB Questions: Transformation
NAME: __________________________
CLASS: _____
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1. Provide AND explain a specific example of a sterile technique protocol that must be followed
during this microbiology lab experiment.
2. List one quantitative and one qualitative observation that will be made during this lab.
(Identify each observation as qualitative or quantitative – AP Biology graders don’t assume
that you know the difference!)
3. What is bacterial genetic transformation?
4. Name the specific organism and two genes involved in this transformation experiment?
5. What is the purpose of heat shock? Use terms: competence, plasmid, transformation, bacteria
3
Lab: Transformation of E. coli with pGLO plasmid
Overview
Plasmids are small circular pieces of DNA often containing extra genes separate from the genome
of a prokaryotic organism. In this lab, you will use a recombinant (transgenic) plasmid to transform E. coli
bacterial cells. You will also calculate the efficiency of this transformation and perform statistical analysis
of your data compared to the class average data.
UNIT 10 CONTENT AND PROCESS-BASED TARGETS:
I: Explain the process of in vivo DNA cloning and its applications for copying eukaryotic
genes in prokaryotic organisms.
V: Describe how the presence of the araC protein promotes positive gene regulation for
the arabinose (Ara) operon of the pGLO plasmid
A: Transform E. coli bacteria with the recombinant DNA plasmid containing the GFP
gene to alter the phenotype of bacteria cells viewed under UV light.
Introduction
The bacterium Escherichia coli (E. coli) is an ideal organism for the molecular geneticist to
manipulate and has been used extensively in recombinant DNA research. It is a common inhabitant of the
human colon and can easily be grown in suspension culture in a nutrient medium such as Luria broth, or in
a petri dish of Luria broth mixed with agar (LB agar) or nutrient agar.
The single circular chromosome of E. coli contains about five million DNA base pairs, only
1/600th the haploid amount of DNA in a human cell. In addition, the E. coli cell may contain small,
circular DNA molecules (1-200kbp) called plasmids, which also carry genetic information. The plasmids
are extra-chromosomal; they exist separately from the chromosome. Some plasmids replicate only when
the bacterial chromosome replicates and usually exist only as single copies within the bacterial cell.
Others replicate autonomously and often occur in as many as 10 to 200 copies with a single bacterial cell.
Certain plasmids, called R plasmids, carry genes for resistance to such antibiotics as ampicillin,
kanamycin, or tetracycline.
In nature genes can be transferred between bacteria in three ways: conjugation, transduction, or
transformation. Conjugation is a mating process during which genetic material is transferred from one
bacterium to another of a different mating type. Transduction requires the presence of a virus to act as a
vector (carrier) to transfer small pieces of DNA from one bacterium to another. Bacterial
transformation involves transfer of genetic information into a cell by direct uptake of the DNA. During
gene transfer, the uptake and expression of foreign DNA by a recipient bacterium can result in conferring
a particular trait to a recipient that previously lacked that trait. Transformation can occur naturally but the
incidence is extremely low and is limited to relatively few bacterial strains. These bacteria can take up
DNA only during the period at the end of logarithmic growth. At this time, the cells are said to be
competent. Competence can be induced in E. coli with carefully controlled chemical growth conditions.
Once competent, the cells are ready to accept DNA that is introduced from another source.
Plasmids can transfer genes (such as those for antibiotic resistance) that occur naturally within
them, or plasmids can act as carriers (vectors) for introducing foreign DNA from other bacteria, plasmids,
or even eukaryotes into recipient bacterial cells. Restriction endonucleases can be used to cut and insert
pieces of foreign DNA into the plasmid vectors. If these plasmid vectors also carry genes for antibiotic
resistance, transformed cells containing plasmids that carry the foreign DNA of interest in addition to the
antibiotic resistance gene can be easily selected from other cells that do not carry the gene for antibiotic
resistance.
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Genetic transformation involves the insertion of some new DNA into the E. coli cells. Plasmid
DNA usually contains genes for more than one trait. Scientists use a process called genetic engineering to
insert genes coding for new traits into plasmids. In this case, the pGLO plasmid carries the GFP gene that
codes for the green fluorescent protein and a gene (bla) that codes for a protein that gives bacteria
resistance to the antibiotic ampicillin. The genetically engineered plasmid can then be used to genetically
transform bacteria to give them these new traits. The real life source of the GFP gene is the
bioluminescent jellyfish Aequorea victoria. Green Fluorescent Protein causes the jellyfish to fluoresce and
glow in the dark. Following the transformation procedure, the bacteria express their newly acquired
jellyfish gene and produce the fluorescent protein, which causes them to glow a brilliant green color under
ultraviolet light.
Several components of the experimental procedure have the function of allowing the plasmids to
enter the bacterial cells. The procedure used to increase the bacterial uptake of foreign DNA is called heat
shock. It is important to follow the directions regarding time. Also important is the RAPID temperature
change and the duration of the heat shock. For optimal results, the tubes containing the cell suspensions
must be taken directly from ice, placed into the water bath at 42oC for 50 seconds and returned
immediately to ice. For example, the absence of the heat shock will result in a 10-fold decrease in
transformants while a 90 second heat shock will give about half as many as would 50 seconds of heat
shock. Transformation solution, CaCl2, is also needed. It is postulated that the Ca2+ cation of the
transformation solution neutralizes the repulsive negative changes of the phosphate backbone of the DNA
and the phospholipids of the cell membrane, allowing the DNA to enter the cells.
The pGLO plasmid that contains the GFP gene also contains the gene for beta-lactamase (bla),
which provides resistance to the antibiotic ampicillin. The beta-lactamase protein is produced and secreted
by bacteria that contain the plasmid. Beta-lactamase inactivates the ampicillin present in the LB nutrient
agar, allowing bacterial growth. Only transformed bacteria that contain the plasmid and express betalactamase can survive on plates that contain ampicillin. Only a very small percentage of the cells take up
the plasmid DNA and are transformed. Untransformed cells cannot grow on the ampicillin selection
plates. pGLO also incorporates a special gene regulation system, which can be used to control expression
of the fluorescent protein in transformed cells. The gene for GFP can be switched on in transformed cells
by adding the sugar arabinose to the cells’ nutrient medium. Selection for cells that have been transformed
with pGLO DNA is accomplished by growth on antibiotic plates. Transformed cells will appear white
(wild-type phenotype) on plates not containing arabinose, and fluorescent green when arabinose is
included in the medium.
Your final task for this investigation will be to learn how to determine the extent to which you
genetically transformed the E. coli cells. This quantitative measurement is called transformation
efficiency. In many experiments, it is important to genetically transform as many cells as possible. For
example, in some types of gene therapy, cells are collected from the patient, transformed in the laboratory,
and then returned to the patient. The more cells that are transformed to produce the needed protein, the
more likely the therapy will work. The transformation efficiency is calculated to help scientists determine
how well the transformation is working.
Procedure – Pre-lab: Failure to complete ANY PORTION OF the pre-lab assignments listed below
(items #1-5) will forfeit your ability to participate in the lab experience for DAY 1!
1. Thoroughly read and annotate the ENTIRE lab handout.
2. Before coming to lab, locate information from a scientific source concerning the concept of sterile
technique (or aseptic technique). Determine how and why sterile technique protocols must be
followed during the transformation experiment to ensure (accurate) results. Please bring a hard copy
of all information found and be able to cite the source of the information using APA format.
3. Complete the data table on page 1 of the lab packet – except for the far right column.
4. Answer the pre-lab “quiz” questions on page 3
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4. Answer the questions below in a (lab) notebook to prepare for lab experience.
A. To genetically transform an entire organism, you must insert the new gene into every cell in the
organism. Which organism is better suited for total genetic transformation: an organism composed
of many cells or an organism composed of a single cell? Explain.
B. Scientists often want to know if the genetically transformed organism can pass its new traits on to
its offspring and future generations. To get this information, which would be a better candidate for
your investigation, an organism in which generation time is minimal or one whose generation time
is long? Explain.
C. Safety is another important consideration in choosing an experimental organism. What traits or
characteristics should the organism have, or not have, to be sure it will not harm the scientist or
the environment? Why?
D. Based on your answers to questions A – C, which would be the best choice for genetic
transformation: a bacterium, an earthworm, a fish, or a mouse? Justify your answer with
evidence.
E. Recall the goal of genetic transformation is to change an organism’s traits (phenotype). Before
any change in the phenotype of an organism can be detected, a thorough examination of its
natural (pre-transformational) phenotype must be made. Observe the colonies of E. coli on your
starter plate. List all observable traits or characteristics that can be described. Obtain additional
observations from at least one other group. Possible qualitative and quantitative observations
include:
a. Number and color of colonies; distribution of colonies on the plate
b. Size of the largest colony, smallest colony, majority of colonies. (units?)
c. Visible appearance when viewed under UV light
d. The ability of cells to live/reproduce in the presence of antibiotic such as ampicillin.
e. Draw the starter plate to describe colony size and distribution.
F. Describe how you could use two LB/agar plates, some E. coli and some ampicillin to determine
how E. coli cells are affected by ampicillin.
G. What would you expect your experimental results to indicate about the effect of ampicillin on E.
coli cells?
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Procedure - Day 1
The transformational procedure involves three main steps. These steps are intended to introduce the
plasmid DNA into the E. coli cells and provide an environment for the cells to express their newly
acquired genes.
To move the pGLO plasmid DNA through the cell membrane you will:
1. Use a transformation solution of CaCl2 (calcium chloride)
2. Perform heat shock to induce competence of E. coli cells
For transformed cells to grow in the presence of ampicillin you must:
3. Provide bacteria with sugar, amino acids, and other monomer units along with a short
incubation period to begin expressing their newly acquired genes
1. Obtain two microtubes. Label them “+pGLO” and “-pGLO.”
2. Open the tubes and using a sterile transfer pipette, transfer 250uL of transformation solution (CaCl2).
Tranformation solution = CaCl2
3. Put the tubes on ice.
4. Use a sterile loop to pick up a single colony of bacteria from your starter plate. Pick up the “+pGLO”
tube and immerse the loop into the transformation solution at the bottom of the tube. Spin the loop
between your index fingers and thumb until the entire colony is dispersed in the transformation
solution (with no floating chunks). Place the tube back in the tube rack in the ice. Immediately place
used loop into the bleach solution in a beaker. Using a new sterile loop, repeat for the “-pGLO” tube.
7
5. Examine the pGLO plasmid DNA solution with the UV lamp. Does the plasmid ‘glow green’ if the
DNA is not inside a cell? Immerse a new sterile loop into the plasmid DNA stock tube. Withdraw a
loopful of plasmid solution. There should be a film of plasmid solution across the ring. (The
appearance is similar to seeing a soapy film across a ring for blowing soap bubbles.) Mix the loopful
into the cell suspension of the +pGLO tube. Close the tube and return it to the rack on ice. Also close
the -pGLO tube. Do not add plasmid DNA to the -pGLO tube. (Why?)
6. Incubate the tubes on ice for 10 minutes. Make sure to push the tubes all the way down in the rack so
the bottoms of the tubes stick out and make contact with the ice.
7. While the tubes are sitting on ice, label your three agar plates on the bottom where the agar is located
(not on the ‘empty’ lid). Label plates with your group name, date, & period number. Write small on the
perimeter on the bottom of dish where the agar is located.
8. Heat shock: Using the foam rack as a ‘float’, transfer both (+) and (-) pGLO tubes into the water bath,
set at 42oC, for exactly 50 seconds. Make sure to push the tubes all the way down in the rack so the
bottom of the tubes stick out and make contact with the warm water. When the 50 seconds are done,
place both tubes directly into ice (out of foam float). For the best transformation results, the change
from the ice (0oC) to 42oC and back to ice must be rapid and timing must be precise! Incubate tubes on
ice for 2 minutes following heat shock.
8
9. Remove the rack containing the tubes from the ice and place on the bench top. Open a tube and, using
a new sterile pipette, add 250 uL of LB nutrient broth to the tube and close it. Repeat with a new sterile
pipette for the other tube. Allow the tubes to “rest” for 10 minutes at room temperature.
10. Tap the closed tubes with your finger to mix. Using a new sterile pipette for each tube, pipette 100uL
of the transformation and control suspensions onto the appropriate plates.
11. Use a new sterile loop for each plate. Spread the suspensions evenly around the surface of the agar by
quickly & gently skating the flat surface of a new sterile loop back and forth across the agar’s surface.
12. Stack up your plates (including E. coli starter plate), making sure the agar is located in the same
direction for all plates, and tape them together. Put your group name and class period on the bottom of
the stack. Your teacher will place the stack upside down (agar-side up) in the 37oC incubator until the
next day. (Incubate plates at 27oC if leaving cultures over a weekend).
9
Procedure - Day 2
After the plates have incubated for 24 hours (or more), address the following with respect to the
experimental results for the three plates: +pGLO LB/amp, -pGLO LB, -pGLO LB/amp.
1. Carefully observe and draw what you see on each of the plates. Be sure your data table allows
comparison between the +pGLO transformed E. coli cells and the non-transformed E. coli cells
(-pGLO). Record the following information in the data tables on pages 1-2 for each plate:
a. How much bacteria growth do you see on each plate? (Count # colonies or estimate %
of plate covered by a bacterial lawn)
b. What color are bacteria under normal room lights and under UV light?
c. What is the size of the largest and smallest colony?
d. Draw each plate to record colony size & distribution
e. Include information about whether or not the cells are resistant to ampicillin.
2.
Follow the steps (a-c) below to calculate transformation efficiency for the +pGLO LB/amp plate.
Transformation efficiency is a number that represents the total number of bacterial cells that
express the protein, divided by the amount of DNA used in the experiment. It tells us the total
number of cells transformed by one microgram of DNA. To calculate it, we need two numbers:
total number of fluorescent colonies growing on your +pGLO LB/amp plate and total amount of
pGLO plasmid DNA in the bacterial cells spread of the LB/amp/ara plate.
a. Determine the total number of transformed cells:
Count the number of colonies on the +pGLO LB/amp plate. We will assume each colony on
the plate is derived from a single, transformed cell. As individual cells reproduce, more and
more cells are formed and develop into a colony. The most direct way to determine the total
number of cells is to count the colonies on the plate. Record this number.
b. Determine the amount of pGLO DNA in the bacterial cells spread on the +pGLO LB/amp plate.
We need two pieces of information to find out the amount of pGLO DNA in the cells spread
on the LB/amp plate: the total amount of DNA we began with and the fraction of that DNA
(in the bacteria) that actually got spread on the LB/amp/ara plate.
i. Determine the total amount of pGLO plasmid DNA used
DNA (g) = concentration of DNA (g/L) X volume of DNA (L)
In this experiment you used 10L of pGLO at a concentration of .08g/L. This means
that each uL of solution contained .08g of pGLO DNA. Calculate the total amount of
DNA used in this experiment, and record this number.
ii. Determine the fraction of pGLO plasmid DNA (in the bacteria that actually got
spread onto the LB/amp/ara plate.
Since not all the DNA you added to the bacterial cells will be transferred to the agar plate,
you need to find out what fraction of the DNA was actually spread onto the LB/amp/ara
plate. To do this, divide the volume of DNA you spread on the LB/amp/ara plate by the
total volume of liquid in the test tube containing the DNA.
Fraction of DNA used =
Volume spread on LB/amp plate (L)
Total sample volume in the test tube (L)
10
You spread 100L of cells containing DNA from a test tube containing a total volume of
510L of solution. Do you remember why there is 510L total solution? (Look in the
procedure, locate all the steps in which you added liquid to the reaction tube, and add the
volumes. Use the above formula to calculate the fraction of pGLO plasmid DNA you
spread on the LB/amp/ara plate. Record this number.
iii. Determine how many ug of pGLO DNA you spread on the LB/amp/ara plate.
To find this, multiple the total amount of pGLO DNA used by the fraction of pGLO
DNA you spread on the LB/amp/ara plate
pGLO DNA spread (g) = total amount of DNA used (g) X fraction of DNA used
Record this number.
c. Use the calculations you have completed thus far to find the transformation efficiency.
Transformation efficiency =
total number of cells growing on the agar plate
amount of DNA spread on the agar plate
3. Transfer ONE colony from the +pGLO LB/amp plate using a sterile inoculating loop to the new
+pGLO LB/amp/ara plate. Streak from the top to the bottom of the plate while gently twisting the
inoculating loop. Turn the plate 900 and repeat the top to bottom streak technique to spread bacteria
cells throughout the agar surface. CAUTION: DO NOT press the inoculating loop into the agar! DO
NOT create divots in the agar surface!
**If your group does not have any colonies on your +pGLO LB/amp plate, please ask
your teacher from which group you should obtain a transformed E. coli colony.**
4. Label your agar plate on the bottom (not the ‘empty’ lid). Label plate with your group name, date, &
period number. Write small, on the perimeter, on the bottom of the dish where the agar is located.
5. AFTER COLLECTING ALL DATA: Squirt bleach solution into the E. coli starter plate, and –pGLO
plates. Use masking tape to secure each dish closed and dispose of the three dishes into the red
biohazard bag (along with gloves at the conclusion of the day’s work).
6. Give your teacher the groups’ labeled +pGLO LB/amp/ara plate to place in the prep room incubator.
Give your teacher the group’s +pGLO LB/amp plate (to save for comparisons tomorrow).
7. Tell your teacher your group’s transformation efficiency (TE). Your teacher will display all groups TE.
Calculate the class average TE. Use the class average to determine the standard deviation for the class
data. For instructions about how to calculate standard deviation, see data table on page 2, or standard
deviation formula sheet in C.O.B., or classroom laminated formula sheet copy.
11
Procedure – Day 3
1. Carefully observe and draw what you see the +pGLO LB/amp/ara plate. Make a comparison between
the +pGLO LB/amp transformed E. coli cells from yesterday and today’s plate. Record the following
information in the tables on pages 1-2 for each plate:
a. How much bacteria growth do you see on each plate? (Count # colonies or estimate %
of plate covered by a bacterial lawn)
b. What color are bacteria under normal room lights and under UV light?
c. What is the size of the largest and smallest colony?
d. Draw each plate to describe colony size & distribution
e. Include information about whether or not the cells are resistant to ampicillin.
2. Follow the Day 2 procedure directions to calculate the transformation efficiency for the +pGLO
LB/amp/ara plate.
3. AFTER COLLECTING ALL DATA & TAKING PICTURES OF YOUR AMAZING
TRANSFORMED, GREEN-GLOWING CELLS: Squirt bleach solution into +pGLO LB/amp and
+pGLO LB/amp/ara plates. Use masking tape to secure each dish closed and dispose of the two dishes
into the red biohazard bag (along with gloves at the conclusion of the day’s work).
4. Tell your teacher your group’s transformation efficiency (TE). Your teacher will display all groups TE.
Calculate the class average TE. Use the class average to determine the standard deviation for the class
data. For instructions about how to calculate standard deviation, see data table on page 2, or standard
deviation formula sheet in C.O.B., or classroom copy.
Post-Lab Analysis Questions: The answers to these questions are not going to be
collected graded. However, the unit exam will require you to write a summary of the lab
methodology and procedure with explanation.
1. If the genetically transformed cells have acquired the ability to live in the presence of the antibiotic
ampicillin, then what might be inferred about the other genes on the plasmid that were used in the
transformation procedure?
2. From the results you obtained, how could you prove that the changes that occurred were actually due to
the procedure that you performed, and not to some other factor?
3. Why did the DNA plasmid itself not glow when exposed to UV light (Day 1, procedure step 5), but
bacterial colonies containing the plasmid do glow (Day 3, step 1)?
4. What factor MUST be present in the bacteria’s environment (agar medium) for the bacteria to express
the green fluorescent protein (GFP)?
5. What is the selective advantage for the bacteria to be able to turn “off” the gene for GFP when the
supply of arabinose is diminished?
6. A scientist conducts as transformation experiment. The relevant information from her procedure and
results are listed below. Use this information to calculate the transformation efficiency. Show all work
and include all units.
DNA plasmid concentration: 0.08 ug/uL
250 uL CaCl2 transformation solution
10 uL pGLO plasmid solution
250 uL LB broth
100 uL cells spread on agar
227 Transformed colonies
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