Chapter 20: Biotechnology
The questions, readings, simulations, and activities contained herein are meant to familiarize you
with key biotechnology terminology and practices that could be covered on the AP biology
exam. The benefit and understanding you gain from these assignments will be commensurate
with the level of effort you put into completing the work. Some class and laboratory time may
be devoted to exploring specific biotechnology concepts, however, you are responsible for
completing all the work contained in this packet. A summative assessment will follow your
completion of this packet to test your knowledge about biotechnology.
Overview
This packet is organized into the following content areas:
1.
2.
3.
4.
5.
6.
7.
8.
What is biotechnology?
Restriction Enzymes
Plasmid-based Transformation
Polymerase Chain Reaction (PCR)
Gel Electrophoresis
Genetically modified organisms (GMOs) – plants and animals
Cloning
Pharmaceuticals (insulin)
1. What is Biotechnology?
Biotechnology - the manipulation (as through genetic engineering) of living organisms or their
components to produce useful usually commercial products (as pest resistant crops, new bacterial
strains, or novel pharmaceuticals); also: any of various applications of biological science used in
such manipulation.
It is important to understand the meaning of the three terms in bold to start this chapter.
Please define.
recombinant DNA – a DNA molecule made in vitro with segments from different sources
genetic engineering – the direct manipulation of genes for practical purposes
gene cloning – the production of multiple copies of a gene
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2. Restriction Enzymes
Read the description of restriction enzymes on page 398 carefully. Label the diagram below
and write the 3 steps involved to make recombinant DNA. See Text for labeling
1. Restriction enzymes
cut the sugar phosphate backbones
at each arrow
2. DNA fragment from another source
is added. Base paring of sticky ends
produces various combinations
3. DNA ligase seals the strands
Questions to consider related to restriction enzymes:
a. Why do DNA molecules always yield the same set of restriction fragments when exposed
to the same restriction enzymes? Because that particular set of enzymes only recognize
certain cut sites that do not change unless mutation occurs.
b. When a DNA fragment has been removed by restriction enzymes why are the ends on the
fragment and the original DNA called “sticky”? Explain.
These fragments have unpaired bases on their ends that can bond to another piece of
DNA.
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c. What enzyme permanently joins a fragment from one DNA molecule to another forming
recombinant DNA? What other role does this enzyme play in DNA replication?
Ligase. It joins Okazaki fragments
d. Predict why restriction enzymes play such an important role in gene cloning.
Enzymes make it possible to remove pieces of DNA from an organism’s genome. When
the same enzyme is used in both organisms the DNA from one can be inserted into
another.
e. The restriction site for an enzyme called PvuI is the following sequence:
5’-CGATCG-3’
3’-GCTAGC-5’
Staggered cuts are made between the T and C on each strand. What type of bonds are
being cleaved? covalent Sugar-Phosphate bonds
f. One strand of a DNA molecule has the following sequence: 5’CCTTGACGATCGTTACCG-3’. Draw the other strand. Will PvuI cut this molecule?
If so, draw the products.YES
CCTTGACGATCGTTACCG
GGAACTGCTAGCAATGGC
3. Plasmid-based transformation
Look at Figure 20.4 on page 399 of your textbook. This is a more detailed discussion of the gene
cloning procedure shown in figure 20.2 of your text. The questions that follow pertain to how a
bacterium can be used to clone genes of interest from another organism.
Please define:
Plasmid – a small circular double stranded DNA molecule that carries accessory genes separate
from those of a bacterial chromosome (also in yeast an some eukaryotes)
Cloning Vector- in genetic engineering a DNA molecule that can carry foreign DNA into a host
cell and replicate there (e. g. plasmid)
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Bacterial transformation – Bacterial cells take up the recombinant plasmid or other DNA
molecule by transformation
a. Explain why the plasmid is engineered with ampR and lacZ?
ampR makes the E. coli resistant to the antibiotic ampicillin, thus only those that took up
theis gene will grow on agar treated with ampicillin. The LacZ is a gene that can
hydrolyze X-gal to form a blue product.
b. After transformation has occurred why are some colonies blue?
These colonies have the lacZ gene intact and did not take up the recombinant DNA so
they can hydrolyze X-gal turning them blue (indicated non-recombinant)
c. Why are some colonies white? Why is this important?
They cannot hydrolyze X-gal because the lacZ is disrupted with recombinant DNA.
d. Why does the lacZ gene not work properly in the recombinant plasmids?
It’s disrupted by the hummingbird gene
e. What benefits are realized by researchers when genes can be easily cloned using
plasmids?
Genes of interest that make a particular protein can be copied in mass and their protein
product could be made.
Plasmid transformation for producing transgenic organisms
Transgenic organism – organisms that contain genes from other species produced by insertion
of recombinant DNA into the genome of a host organism
Transgenic Plants – transformed by using bacteria such as Agrobacterium tumefaciens,
removing the cell wall or directly injected
Transgenic Animals – transformed by injecting DNA directly into the nucleus of egg cells.
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Read the description of Genetic Engineering in Plants on page 421 and 422 in your textbook.
Label Figure 20.25 below and write the 3 steps involved to make a transgenic plant.
Step 1 – Ti plasma is isolated from
Agrobacterium tumefaciens
Step 2 – Foreign gene of interest is
Inserted into the Ti plasmid
(recombinant plasmid)
Step 3 - Return plasmid to
agrobacterium and spray it onto
the plant as a liquid suspension
infecting the plant. Plant integrates
plasmid DNA into chromosomal DNA.
Now, make your own transgenic plant. Follow the link below and follow the directions to make
a transgenic tomato plant that is both herbicide and pest resistant.
PBS - Transgenic Manipulation
Answer the questions below pertaining to the simulation:
1. What is Bt and where did it come from?
A bacterium called Bacillus Thuringiensis that produces toxins that are harmful to insects
but not to mammals.
2. What transgenic properties will your tomato plant have?
It will be resistant to insect pests and also be resistant to herbicides
3. What do you think the “vector” is in the simulation?
The vector is a plasmid
4. Although not shown what other gene was added to the vector?
A gene that confers herbicide resistance to the plant
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5. How did the Bt gene and herbicide resistance gene get incorporated into the genome of
the tomato plant cells?
The agrobacterium cell which contains the vector with Bt and herbicide resistance genes
is capable of infecting the plant cells and inserting the vector into the plant cell genome.
6. In this simulation how was it confirmed that the plant cells have taken up the desired
genes?
The plant cells were sprayed with a herbicide, only those plants that have both herbicide
resistance and Bt will survive.
7. Explain from a practical standpoint how this tomato plant would increase a farmers yield
(your tomato seed would be called a “stacked” variety because it is modified in two
ways).
The farmer could spray for weeds without having to worry about damaging the crops. A
farmer could get more yields because less is loss to insect damage. Plants may grow
bigger and produce more fruit because the plant is not being stressed by weed or insect
pests.
8. Imagine you are the biotechnology company that developed this transgenic tomato plant,
what risks do you face after the seeds are sold and how will you protect your technology?
Since seeds generated from the transgenic plants will also have transgenic properties the
seeds could be saved circumventing the need for purchasing the seeds. Produce seeds
that only produce infertile offspring.
4. Polymerase chain reaction (PCR)
PCR is a Nobel Prize–winning idea that is used by scientists to amplify DNA, particularly when
the quantity of DNA is very small or contaminated. PCR is discussed on pages 403 and 404 of
your textbook.
Use the following link to watch the 3D and 2D animation of PCR in action under the heading
“Technique”:
Polymerase Chain Reaction (PCR) Collection: DNA Learning Center
a. Explain the three initial steps that occur in cycle 1 of PCR (use figure 20.8).
Heating and denaturing to separate the DNA, Annealing primers to the DNA target
sequence at a lower temperature, and extending the DNA by polymerase adding
nucleotides to the primers at the 3’ end.
b. According to your text what was the “key” to automating PCR?
A heat stable DNA polymerase
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c. How many cycles does it take to get the actual target sequences of DNA made?
3 cycles
d. What four “ingredients” must be brought together in the reaction to make sure that PCR
works?
Nucleotides, Primers, DNA Polymerase, Target Sequence
e. What biotechnology process discussed earlier in this assignment could be bypassed if
PCR were used instead?
Gene cloning using plasmids
5. Gel Electrophoresis
Gel Electrophoresis was commonly called “DNA Fingerprinting”. Bear in mind that this
technique has nothing to do with actual fingerprints, although it could be used to analyze DNA
samples from a crime scene and potentially identify or rule out a possible suspect. This type of
analysis is now called “DNA profiling” or “DNA testing”.
Use the link below to conduct your own virtual Gel Electrophoresis analysis:
University of Utah - Gel Electrophoresis
Questions from simulation.
a. Scientists use gel electrophoresis to sort DNA according to what factor?
The length of the DNA fragment
b. Why is the DNA sample to be separated by gel electrophoresis always loaded at the
cathode or negative end of the power source?
DNA carries negative charges on phosphate groups and is attracted to positive
c. Shorter strands will move faster or slower through the gel?
Faster
d. Why is a salt water solution used to make the gel and used as a buffer in the gel box?
Conducts electrical current
e. Why is a loading buffer used in combination with the actual DNA samples?
The loading buffer contains dye (allows progress of band migration to be seen) and
sucrose (makes the sample dense so it sinks into the well).
f. What provides the proof that electrical current is actually running through your gel and
buffer?
Bubbles will form at the cathode and anode ends of the chamber
g. What are the DNA bands in the gel stained with so that they can be seen, and what type
of light will they be exposed to?
Keeps DNA from running off the gel (indicator). Exposed to UV light.
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h. What does bp stand for? Record your DNA sample bp estimates here: ___________
_____Base________
___Pairs__________
Additional Questions:
How did the DNA sample get cut into fragments to be separated by gel electrophoresis in the
first place?
Restriction enzymes cut the DNA into fragments
If our DNA is 99.9% the same from one human to the next, why would DNA profiling work to
distinguish one person’s DNA from another? Explain. (hint: check out “Can DNA Demand a
Verdict” link under the simulation)
It turns out that even though we only have .1% difference between individuals the sheer number
of DNA bases pairs (3 billion) still provides 3 million differences between people.
What are short tandem repeats (STRs) and how are they used in DNA profiling?
Tandemly (consecutive) repeated units of 2 to 5 base sequences, that are highly variable from
person to person
For example: ACAT – 18 repeats in one person, only 3 repeats in another
Use your text page 406 for this question:
To the right of the β-globin alleles, draw a gel showing the different pattern obtained from a
normal patient and a sickle-cell patient. For help, examine Figure 20.10 from your text. This is
described in your text, please note that the gel results show a normal and mutant band pattern on
the gel. Normal makes three bands and mutant (sickle) makes two. This concept hinges on the
fact that restriction enzymes may not recognize a cut site if a mutation occurs resulting in fewer
fragments (same as our cystic fibrosis lab).
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Complete the flowchart about how DNA fingerprints are made.
Restriction enzymes
are used to cut the DNA into fragments
containing genes and repeats.
The restriction fragments are separated according to size using gel
electrophoresis .
The DNA fragments containing repeats are then labeled using
radioactive probes
. This labeling produces a series of
bands—the DNA fingerprint.
Study the DNA fingerprint below. Which two samples may be from a set of identical twins? How
do you know? Sample 1 and Sample 4 – banding patterns match
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6. Genetically Modified Organisms (GMO) - plants and animals
Have you eaten genetically modified (GM) foods this week? Probably. The majority of GM
organisms that contribute to our food supply are not animals, but crop plants.
GM Crops – transgenic plants that resist pests, herbicides, disease and result in increased yields.
-Use of these crops is on the rise
-Introduced in 1996 (Round Up resistant soybean)
-As of 2014 GM crops made up 94% of soybeans, 96 % of cotton and 93% of corn
Examples: Roundup ready soybeans, Bt corn, tomatoes, rice, and potatoes
Pick one of the GM plants below and do some research related to the following:
1. What makes it GM/transgenic?
2. What other organism was used as a source of the inserted/manipulated gene?
3. What process is commonly used to accomplish the modification?
4. What benefits does the transgenic plant promise or deliver?
5. Are there any concerns or drawbacks associated with using the GMO?
List of plant GMOs:
Herbicide tolerance – soybeans
Insect resistance – corn
Altered fatty acid composition – canola
Vitamin Enrichment – rice
Vaccines – tobacco
Oral vaccines - maize
Record your answers to the questions above below:
1. The corn plant received a gene from a bacterium
2. A soil bacterium called Bacillus Thuringiensis (Bt)
3. The Bt gene is removed from the bacillus thuringiensis bacterium. It is then inserted into
a plasmid that is taken up by agrobacterium. This agrobacterium infects corn plant cells
and inserts the Bt gene into the genome of the corn plant. Corn plants are cultured from
the BT modified cells, and the plant then has the desired transgenic properties.
4. The Bt corn plant will kill the European corn borer a major pest that infests the ears of
corn.
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GM animals – engineered to increase production, nutritional benefit or product not typically
associated with that animal.
30% of milk in US is coming from cows injected with bovine growth hormone (BGH)
Examples of GM foods from animals:
-Cows – rBGH, increased milk output
-Pigs – leaner meat, omega 3 fatty acids
-Goats – spider genes to manufacture silk, antibacterial goat milk, proteins, drugs
-Salmon – growth hormone, shorter time to market
Aqua Bounty Farms - GM Salmon – use this link to watch a short video about Aqua Bounty and
their 25 year fight to get their genetically modified salmon approved by the FDA. Please answer
the questions below:
a. What other fish was used to make the Atlantic salmon transgenic?
Ocean pout
b. What does the gene from the other fish change about the Atlantic salmon?
It cause the salmon to grow year round greatly increasing it’s growth rate, but not overall
size
c. If the transgenic salmon doesn’t grow bigger overall what is the advantage claimed by
Aqua Bounty?
The time required to raise a salmon for market is cut in half.
d. What is the “good genes” hypothesis mentioned by the researcher?
Larger fish attract more mates, so the genes from larger salmon are passed on more
frequently than those of smaller fish. (concern is that large GM salmon could affect
breading populations if they were to escape and mingle with wild populations)
e. Why does the Aqua Bounty claim that even if a GM salmon were to escape from a fish
pen at sea that it would have no impact on wild salmon populations?
Aqua Bounty fish are all females and they have all been engineered to be sterile.
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AquaBounty's salmon (rear) have been genetically modified to grow to market size in about half the
time as a normal salmon — 16 to 18 months, rather than three years.
The rest of the story: Genetically Modified Salmon Is Safe to Eat, FDA Says: NPR
Follow the link above and read through the latest news about GM salmon.
Write one paragraph below about your opinion on the issue of GM salmon. What points
do you agree with, what do you disagree with? Would you personally eat salmon that is
transgenic? What are the possible benefits of GM salmon as a food source for others
around the world? Are you concerned about possible ecological threats?
I already prefer salmon from Giant that is farm raised w/color added (boneless of course).
So I say bring on the GMO salmon!
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7. Cloning
Clones are organisms that are exact genetic copies of each other. This can happen naturally as in
the case of identical twins or artificially using laboratory techniques. Organismal cloning is the
practice of cloning whole multicellular organisms from single cells. Current interest in
organismal cloning has been generated because of the potential to generate stem cells.
Let’s examine organismal cloning methods before exploring types of stem cells. Follow the link
below to the University of Utah’s cloning web page.
University of Utah – Cloning
Click on the tab “What is Cloning?”
Two types of cloning are discussed on this page. Before describing both types of cloning, what
was the most famous animal cloned, and in what year was it cloned?
Briefly summarize each method of organismal cloning in the space below:
Embryo Twinning- in a petri dish an early embryo is separated into individual cells and is
allowed to divide and develop for a short time. These cells are then inserted into a surrogate
mother where they finish development. The offspring are identical because each cell came from
the same zygote.
Somatic Cell Nuclear Transfer- Essentially an enucleated egg cell has it’s nucleus replaced
with a somatic cell from another member of the population that is the same species, but unrelated
(in other words, egg donor and nucleus donor are different organisms). Once the nucleus from
the somatic cell is in the egg (usually electric shock is used to fuse the membranes together), the
egg cell is encouraged to divide, then is placed in the uterus of a surrogate mother.
Complete the diagram below that details the cloning of a sheep. (top to bottom: somatic cell,
nucleus, divide, blastocyst/embryo
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Please return to the Univ. of Utah cloning website and click on “Click and clone”, here you will
simulate the cloning of a mouse named Mimi. Work through the simulation to become familiar
with the process.
Again, return to the Univ. of Utah cloning website and click on “Why Clone?”
List the six way that cloning may be useful.
a. _____Medicine___________________
b. ______Reproduce a deceased pet__________________
c. ______rare or extinct species__________________
d. _____livestock___________________
e. _____drug production___________________
f. ______Human cloning__________________
Briefly explain the medical reasons for cloning.
Stem cells build, maintain and repair the body. Stem cells from an individual could be
cloned for and made identical to that individual avoiding possible rejection.
Cloning animals used in research that may already contain mutations of interest or conferred
properties from genetic engineering could be easily produced so the research could be sped
up by not having to raise and modify the research animals from scratch.
Give at least two reasons why a clone might not be a carbon copy of the donor organism.
Changes in the environment of the clone may effect behavioral changes in the clone (nature
vs. nurture argument)
X-inactivation and formation of barr bodies
What are some barriers to human cloning, both technical and ethical?
Ethically there are questions related to what purposes justify cloning, and how cloned
individuals may be treated. Technically the technology is not sound enough to successfully
clone a human, success rate is very low and most pregnancies end in miscarriages.
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Use your text to answer the following questions (pgs. 412-416)
1. What is a totipotent cell?
Cells capable of developing into all the different cell types of that species (usually these
cells only remain totipotent up to the 16 cell stage in mammals)
2. What are stem cells?
Cells that are undifferentiated and are capable of reproducing indefinitely and
differentiating into specialized cells of one or more types
3. What is the difference between embryonic stem (ES) cells and adult stem cells?
ES cells are highly variable in that they can be induced to differentiate into almost any
type of specialized cell
Adult stem cells although present in animal tissue are not able to give rise to all cell
types, but can still specialize depending on where the cells are found (bone marrow adult stem cells can differentiate into all the types of blood cells)
4. What are pluripotent cells?
Cells capable of differentiating into many different cell types (embryonic stem cells)
5. Why might induced pluripotent stem cells (iPS) resolve the debate about using stem cells
for medical treatments?
Adult stem cells are coaxed back in time by using retroviruses to insert “stem cell”
master regulatory genes that turn the adult stem cell into an ES cell (these iPS cells do
everything ES cells do). No embryonic tissue is used, circumventing the embryo stem
cell ethical debate.
8. Pharmaceuticals (insulin)
The pharmaceutical industry has been revolutionized by advances in biotechnology. The last
part
of this assignment involves you completing a case study from the National Center for Case Study
Teaching in Science. The case study is entitled From Cow Juice to a Billion Dollar Drug, with
Some Breakthroughs in Between. This case chronicles the science and methodology involved in
identifying and manufacturing insulin for the treatment of type I diabetes.
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