AP Biology: Laboratory
Gene Transformation
Model 1
Genetic transformation occurs when a cell is purposely modified to “take up” and express a new piece of
genetic material (DNA) from another organism. Genetic transformation literally means change caused by genes,
and it involves the insertion of a new gene into an organism’s existing DNA in order to change the organism’s
traits. Remember: A gene is a piece (or section) of DNA which provides the instructions for making (coding
for) a particular protein.
Genetic transformation is used in many areas of biotechnology. In agriculture, genes coding for traits such as
frost, pest or drought resistance can be genetically transformed into plants. In bioremediation, bacteria can be
genetically transformed with genes enabling them to digest oil spills. In medicine, diseases caused by defective
genes are beginning to be treated by gene therapy; that is, by genetically transforming a sick person’s cells with
healthy copies of the gene involved in their disease.
Bacteria commonly undergo the process of transformation naturally. In addition to one large circular
chromosome, known as a nucleoid, which contains all of the genes a bacterium needs for its normal existence,
bacteria naturally contain one or more tiny circular pieces of DNA called plasmids. Plasmid DNA contains
genes for traits that may be beneficial to bacterial survival under certain environmental conditions. In nature,
bacteria can transfer plasmids back and forth and integrate them into their nucleoid, allowing them to share
these beneficial genes. This mechanism allows bacteria to adapt to new environments. Figure 1 displays a
model of a bacterial cell with a nucleoid and plasmid before and after plasmid integration.
Plasmid
Nucleoid
Figure 1
1. What is a plasmid and how are plasmids different from a bacterium’s nucleoid (primary DNA strand)?
2. Compare the size of plasmids and nucleoids in a bacterial cell.
3. In order for bacterial DNA to integrate a plasmid, bacteria often use restriction enzymes. Propose an
explanation for why restriction enzymes would be useful in this process.
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AP Biology: Laboratory
4. Bacteria commonly contain plasmids that code for genes that alter the structure of the bacterial cell wall,
making the cell resistant to specific antibiotics.
a. How is the presence of this plasmid beneficial to bacteria?
b. What would be expected to occur if such a plasmid was removed from a bacterium exposed to
antibiotics?
Model 2
A group of students performed a bacterial transformation using a plasmid called pGLO. The pGLO plasmid
contains the Green Fluorescent Protein (GFP) gene that comes from the bioluminescent jellyfish Aequorea
victoria. This gene allows the jellyfish to produce a protein that fluoresces, or glows, under ultraviolet light.
This gene can be “turned on” in transformed cells by simply adding the sugar, arabinose, to the bacterial cells’
nutrient medium known as agar. Bacterial colonies, which normally appear as white, will then appear green
under a UV light. Additionally, pGLO contains the gene amp for resistance to the antibiotic ampicillin.
Bacteria are normally killed by this antibiotic, but the presence of the amp gene allows for growth, even when
ampicillin is present in the environment.
To move the plasmid through the E. coli cell membrane the students immersed the bacteria in a solution of
calcium chloride (CaCl2) and “heat shocked” the mixture (move it from cold to hot conditions quickly) which
opens small pores in the bacteria allowing the transformed plasmid to move into the bacterial cells.
5. The pGLO plasmid carries two genes. Draw a model of the pGLO plasmid, including the two genes that it
contains. Describe the function of each gene.
6. What relationship does the sugar arabinose have to the function of pGLO?
7. A student argues that because the GFP gene originates from jellyfish (a eukaryote), it cannot be expressed
in E. coli (a prokaryote) after transformation because the two organisms express genes in different ways. Is
the student correct? Explain your reasoning.
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AP Biology: Laboratory
8. The students carry out the transformation procedure in some bacterial samples and expose the bacteria to
different treatments. Each treatment is shown below.
Environmental
Condition
Agar
Agar + Arabinose
Agar + Ampicillin
Agar + Arabinose +
Ampicillin
pGLO plasmid
present
a. Which of the environmental conditions listed serves as a control group in this experiment? Explain
your reasoning.
b. For each of the environmental conditions, determine the color (white or green) of the bacterial
colonies that will most likely be seen under UV light if the transformation procedure is successful.
Write these in the table above and explain your predictions in the space below.
9. Transformation is not 100% efficient; in other words, some bacterial cells may take up a DNA fragment
while others do not.
a) Imagine that students performed the transformation procedure and plated the bacteria on agar
containing arabinose only. The students allow the bacteria to grow and observe some white colonies
and some green colonies when observed over ultraviolet light.
 Explain why green colonies are observed.

Explain why white colonies are observed.

A student concludes that, based on this data, the transformation was not 100% efficient. Is the
student’s conclusion correct? Explain your answer.
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AP Biology: Laboratory
b) Based on the information provided, which plates in this experiment are expected to allow growth of
ONLY transformed bacteria? Explain your reasoning.
10. The students also carry out for additional controls of this procedure, growing untransformed bacteria in each
of the environmental conditions. This allows them to ensure that the E. coli is viable (able to grow). For
each of the environmental conditions in the table, determine the following and explain your answer.
 Will bacteria grow?
 If bacteria grow, what color (white or green) will the colonies be?
Environmental
Condition
Agar
Agar + Arabinose
Agar + Ampicillin
Agar + Arabinose +
Ampicillin
pGLO plasmid
absent
11.
Students repeated their experiment, but forgot to immerse the bacterial sample in CaCl2. Would the
outcome of their experiment most likely match the predictions made in question 8 or in question 10? Explain
your reasoning.
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AP Biology: Laboratory
Transformation Pre-lab
The pBLU plasmid contains genes for two proteins: β-galactosidase
and ampr (see figure at right). Β-galactosidase is an enzyme usually
responsible for digesting sugar. However, it is able to digest
substances with similar structures to a sugar, like the chemical Xgal. X-gal is colorless, but when it is cleaved by beta-galactosidase,
one of the products is dark blue. The product therefore makes
bacterial colonies appear blue instead of their usual white color.
Ampr is a protein that confers resistance to the antibiotic ampicillin
so that bacteria can grow even when treated with this antibiotic. E.
coli bacteria will be used in this experiment. The nutrient source
provided so that the bacteria can grow will be Luria broth (LB) in
agar.
Answer the following questions in your laboratory notebook.
1. Read the experimental procedure on pages 7-8.
2. What affect does ampicillin have on E.coli bacteria that do not contain pBLU?
3. What affect does X-gal have on E.coli bacteria that contain pBLU?
4. In this experiment, you will prepare six plates of bacteria, each with different treatments. In the table below,
include the following:
a) Prediction: Will bacteria grow AND if it does grow, what color (white or blue) will it be?
b) Reason: Justify your prediction.
LB
LB + ampicillin
LB + ampicillin + X-gal
Plate 1
Plate 3
Plate 5
w/ Plasmid
Plate 2
Plate 4
Plate 6
w/out Plasmid
5. “Negative controls” in an experiment are control groups for which no phenomenon (in this case, bacterial
growth) is expected. They ensure that there is no effect when there should be no effect. Which plates serve
as negative controls in this experiment? Explain your reasoning.
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AP Biology: Laboratory
6. “Positive controls” in an experiment are control groups for which the phenomenon is expected. They ensure
that there is an effect when there should be an effect. Which plates serve as positive controls in this
experiment? Explain your reasoning.
7. Which plate(s) in this experiment are the only ones expected to allow growth of ONLY transformed
bacteria? Explain your reasoning.
8. Explain the purpose of step 11 in the experimental procedure.
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AP Biology: Laboratory
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AP Biology: Laboratory
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AP Biology: Laboratory
Introduction
1. Explain what transformation is
2. Describe one specific example of how transformation is useful scientific tool
3. Include table of predictions and reasons for each treatment in transformation lab (question 4 of pre-lab)
Methods and Materials
1. Write Bacterial Transformation Procedure was carried out as written on pages 7 and 8 of the laboratory
manual.
Results
1. Design a data table to record your observed results. If your results differed from your predictions, explain
what you think may have occurred.
2. Design a data table to count the number of individual colonies and, using a permanent marker, mark each
colony as it is counted. If the cell growth is too dense to count individual colonies, record “lawn.”
3. Transformation efficiency is expressed as the number of antibiotic-resistant colonies per µg of plasmid
DNA. The object is to determine the mass of plasmid that was spread on the experiment plate and that was,
therefore, responsible for the transformants (the number of colonies) observed.
Because transformation limited to only those cells that are competent, increasing the amount of plasmid
used does not necessarily increase the probability that a cell will be transformed. A sample of competent
cells is usually saturated with the addition of a small amount of plasmid, and excess DNA may actually
interfere with the transformation process.
a. Determine the total mass (in µg) of plasmid used. Remember, you used 10 µL of plasmid at a
concentration of 0.005 µg/ µL.
Total mass = volume x concentration
b. Calculate the total volume of the cell suspension prepared.
c. Now calculate the fraction of the total cell suspension that was spread on the plate.
Volume suspension spread/total volume suspension = fraction spread
d. Determine the mass of a plasmid in the cell suspension spread.
Total mass plasmid x fraction spread = mass plasmid DNA spread
e. Determine the number of colonies per µg plasmid DNA. Express your answer in scientific notation.
Colonies observed/mass plasmid spread = transformation efficiency
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AP Biology: Laboratory
Discussion and Conclusions
Complete the following to earn maximum points in each section:
1. SP 5, Analyze
- Describe the meaning of the transformation efficiency value calculated
2. SP 5, Determine
- Compare hypothesis (question 4 of pre-lab) with observed results (question 1 of results) to explain
whether or not your predictions were supported.
- Determine if transformation occurred in your experiment and explain your reasoning
3. SP 5, Refine
- Discuss sources of experimental error
- Identify one factor that you think would increase transformation efficiency and one factor that would
decrease transformation efficiency. Explain your reasoning.
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