SENIOR HIGH SCHOOL

GENERAL BIOLOGY 2
Quarter 1–Module 1:
Genetic Engineering and
Recombinant DNA Technology

SCHOOL INITIATED MODULE

Gen. Bio. 2 – SENIOR HIGH SCHOOL
Alternative Delivery Mode: Self – Learning Module (SLM)
Quarter 1 – Module 1: Genetic Engineering and Recombinant DNA Technology
First Edition, 2020

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 SHS

GENERAL BIOLOGY 2
Quarter 1 – Module 1:
Genetic Engineering and
Recombinant DNA Technology

SCHOOL INITIATED MODULE

Introductory Message
For the facilitator:

Salutations! Welcome to the Senior High School General Biology 2 Alternative Delivery
Mode (ADM): Self-Learning Module (SLM) on Genetic Engineering and Recombinant DNA
Technology.

This learning module is based from the competency set by the standard of the K to 12
Curriculum. This was carefully written and designed to introduce varied activities for Senior High
School learners with the help of the teachers or the facilitators in supplying answers to all
activities. This emphasizes the basic processes involved in genetic engineering, and the
applications of recombinant DNA technology. This module will be beneficial to the learners and
parents as this showcases interactive tasks for the mastery of the competency. Furthermore,
students may or may not utilize separate sheets in answering the pre-test, self-check exercises
and post-test.

i
 For the learner:

Hi there! Welcome to the General Biology 2 Supplementary Learning Material (SLM)

Module on Genetic Engineering and Recombinant DNA Technology.

The objective of this interactive module is that it will suit your interests and learning
needs. This contains varied activities that will test your skills in following instructions, in reading,
and in writing. You will be starting with a pre-test to assess your prior knowledge on the lesson.
Moreover, the discussions of the lesson are provided and the series of activities are prepared
within the parts of the module. Your outputs on the given activities are needed and are
important for the efficacy of the context and the content of the learning module. Thus, this
module has the following parts and corresponding icons: (STEM_BIO11/12-IIIa-b-6 and
STEM_BIO11/12-IIIa-b-7)

This module has the following parts and corresponding icons:

This will give you an idea of the skills or

What I Need to Know
competencies you are expected to learn
in the module.

This part includes an activity that aims to check

What I Know
what you already knew about the lesson to take. If
you get all the answers correct (100%), you may
decide to skip this module.
This is a brief drill or review to help you link the
What’s In current lesson with the previous one.

In this portion, the new lesson will be introduced to

What’s New
you in various ways such as a story, a song, a
poem, a problem opener,an article, an activity or a
situation.
This section provides a brief discussion of the
What is It
lesson. This aims to help you discover and
understand new concepts and skills.

This comprises activities for independent practice

What’s More
to solidify your understanding and skills of the
topic. You may check the answers to the exercises
using the Answer Key at the end of the module.

This includes questions or blank

What I Have Learned
sentence/paragraph to be filled in to process what
you learned from the lesson.

This section provides an activity which will help

What I Can Do
you transfer your new knowledge or skill into real
life situations or concerns.

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 This is a task which aims to evaluate your level of
Assessment
mastery in achieving the learning competency.

In this portion, another activity will be given to you

Additional Activities
to enrich your knowledge or skill of the lesson
learned. This also tends retention of learned
concepts.

This contains answers to all activities in the

Answer Key
module.

At the end of this module you will also find:

References This is a list of all sources used in developing this module.

The following are some reminders in using this module:

1. Use the module with care. Do not put unnecessary mark/s on any part of the module.
Use a separate sheet of paper in answering the exercises.
2. Don’t forget to answer What I Know before moving on to the other activities included in
the module.
3. Read the instruction carefully before doing any task.
4. Observe honesty and integrity in doing the tasks and checking your answers.
5. Finish the task at hand before proceeding to the next.
6. Return this module to your teacher/facilitator once you are through with it.

If you encounter any difficulties in answering the activities in this module, do not hesitate to
ask assistance to your teacher or facilitator. Always bear in mind that you are not alone.

We certainly hope that through this material, you will experience meaningful learning and
will gain deeper understanding of the relevant competencies. Just have trust yourself and YOU
CAN DO IT!

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 What I Need to Know

You will learn the essential terms commonly used when discussing Genetic Engineering
and Recombinant DNA Technology. This module will walk you through activities and lesson
exercises to better relate to the processes involved in genetic engineering, and discuss the
applications of recombinant DNA technology. This module targets to prepare the 21st -century
learners like you.
In this module you are expected to engage into various activities that will help you
understand the concept of Genetic Engineering and Recombinant DNA technology. You will be
able to do the following:

 outline the processes involved in genetic engineering.(STEM_BIO11/12-IIIa-b-6); and

 discuss the applications of recombinant DNA.(STEM_BIO11/12-IIIa-b-7)

What I Know

To gear you up on this lesson, let us test your prior knowledge on genetic engineering
and recombinant DNA technology. Do not worry about your score since this will only serve as
your basis in delving deeper into the processes involved in genetic engineering and applications
of recombinant DNA technology.

Directions: Read each statement and choose the letter of the correct answer and write it on
your answer sheets.

1. In the reproductive cloning of an animal, the genome of the cloned individual comes from?
A. an egg cell C. a sperm cell
B. A body cell D. any gamete cell
2. What carries a gene from one organism into a bacteria cell?
A. a plasmid C. an electrophoresis gel
B. a restriction enzyme D. polymerase chain reaction
3. What is a genetically modified organism?
A. a hybrid organism
B. a plant with certain genes removed
C. an organism with an artificially altered genome
D. any agricultural organism produced by breeding or biotechnology
4. What type of insulin is produced by recombinant DNA technology?
A. A combination of E. coli and human insulin
B. Engineered to be more effective than human insulin
C. Identical to human insulin produced in the pancreas
D. Cheaper but less effective than pig insulin for treating diabetes

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5. What is recombinant DNA?
A. DNA that has been sequenced
B. DNA that causes genetic disorders
C. Adding DNA from one organism into the DNA of another
D. DNA which has been changed over generations by natural selection
6. It is the process of making changes in the DNA code of a living organism?
A. inbreeding C.selective breeding
B. hybridization D.genetic engineering
7. What is the ultimate source of genetic variation?
A. radiation C. inbreeding
B. mutations D. hybridization
8. It refers to the entire collection of genes within human cells is referred to as the?
A. pedigree C. gene map
B. karyotype D. human genome
9. Which of the following enzymes in bacteria are responsible for restricting the growth of
viruses?
A. Gyrase C. topoisomerase
B. protease D. restriction endonuclease
10. Which enzyme is used to join together two different types of DNA molecules?
A. ligase C. exonuclease
B. protease D. endonuclease

Lesson

1 Genetic Engineering

As you continue your quest, your goal in this section is to outline the processes involved
in genetic engineering. The central dogma of molecular biology explains the flow of gene
information from genes to protein. It provides a molecular mechanism in understanding how
genotype translates to phenotype. It became apparent then that changing an organismal trait is
possible by altering its genetic makeup. Genetic engineering involves manipulating genes in
order to make useful products. This part describes the techniques used and discusses different
applications of genetic engineering.

What’s In

Genetic engineering involves the artificial manipulation, modification, and recombination

of DNA or other nucleic acid molecules in order to alter an organism or population of organisms.
The term genetic engineering initially referred to various techniques used for the modification or
manipulation of organisms through the processes of heredity and reproduction. As such, the
term embraced both artificial selection and all the interventions of biomedical techniques among

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them artificial insemination, in vitro fertilization(e.g.,“test-tube”babies), cloning, and gene
manipulation.

Many people are unaware that humans have been practicing genetic engineeering since
ancient times. Moreover, genetic engineering is involved in classical plant breeding which uses
deliberate interbreeding(crossing) of closely or distantly related individuals to produce new crop
varieties or lines with desirable properties. Plants are crossbred for example to introduce
traits/genes from one variety or line into a new genetic background. This type of modification is
of great help to different aspects of production such as in agriculture and food production.

Selective breeding in agricultural crops and livestock has actually altered the genetic
makeup of these organisms over the centuries in such a way that they no longer resemble their
non-domesticated relatives. This practice has been common long before genes were discovered.
In artificial selection, however, genes are only manipulated indirectly because it is only the
physical traits that are selected. Most often, a gene from another species is added to an
organism's genome to give it a desired phenotype which is beneficial and useful.

With the advancement of technology, scientists are now able to directly study and
manipulate genes in the lab. This requires isolating a specific gene and making several copies
of that gene, a process called DNA cloning. To get multiple copies of a gene or other piece of
DNA you must isolate, or ‘cut’, the DNA from its source and then ‘paste’ it into a DNA vector that
can replicate (or copy) itself.

The four main steps in DNA cloning are:

Step 1. The chosen piece of DNA is ‘cut’ from

the source organism using restriction enzymes.

Step 2. The piece of DNA is ‘pasted’ into

a vector and the ends of the DNA are joined
with the vector DNA by ligation.

Step 3. The vector is introduced into a host cell,

often a bacterium or yeast, by a process
called transformation. The host cells copy the
vector DNA along with their own DNA, creating
multiple copies of the inserted DNA.

Step 4. The vector DNA is isolated (or

separated) from the host cells’ DNA and purified.

DNA that has been ‘cut’ and ‘pasted’ from an

organism into a vector is called recombinant
Figure 1. Cloning into a Plasmid
DNA. Because of this, DNA cloning is also called
recombinant DNA technology. www.davidson.bio.edu

What is cloned DNA used for?

DNA cloning is used to create a large number of copies of a gene or other piece of DNA.
The cloned DNA can be used to:

 Work out the function of the gene

 Investigate a gene’s characteristics (size, expression, tissue distribution)
 Look at how mutations may affect a gene’s function
 Make large concentrations of the protein coded for by the gene

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 Figure 2. An illustration of how cloning works using a sheep (Image from: www.slideshare.net).

What other types of cloning are there?

The term ‘cloning’ is also used to describe other laboratory processes:

 Reproductive cloning is the process of making a genetically identical copy of an

organism.
 Therapeutic cloning is the process of making multiple copies of a cell to treat a disease.v

Key Points:

 Genetic engineering involves manipulating genes in order to make useful products. This
includes the use of molecular techniques to modify the traits of a target organism. The
modification of traits may involve:

1. Introduction of new traits into an organism.

2. Enhancement of a present trait by increasing the expression of the desired gene.

3. Enhancement of a present trait by disrupting the inhibition of the desired genes’

expression.

What’s New

Scientists have developed a new technique to genetically engineer bacteria by making

human-made DNA invisible to a bacterium's defenses. In theory, the method can be applied to
almost any type of bacteria. Below is an Article published on May 28, 2019 on Science Daily
from Forsyth Institute.

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 Bacteria are everywhere. They live in the soil and water, on our skin and in our bodies.
Some are pathogenic, meaning they cause disease or infection. To design effective treatments
against pathogens, researchers need to know which specific genes are to blame for
pathogenicity. Scientists can identify pathogenic genes through genetic engineering. This
involves adding human-made DNA into a bacterial cell. However, the problem is that bacteria
have evolved complex defense systems to protect against foreign intruders -- especially foreign
DNA. Current genetic engineering approaches often disguise the human-made DNA as
bacterial DNA to thwart these defenses, but the process requires highly specific modifications
and is expensive and time-consuming.

In a paper published recently in the Proceedings of the National Academy of

Sciences journal, Dr. Christopher Johnston and his colleagues at the Forsyth Institute describe
a new technique to genetically engineer bacteria by making human-made DNA invisible to a
bacterium's defenses. In theory, the method can be applied to almost any type of bacteria.
Johnston is a researcher in the Vaccine and Infectious Disease Division at the Fred Hutchinson
Cancer Research Center and lead author of the paper. He said that when a bacterial cell
detects it has been penetrated by foreign DNA, it quickly destroys the trespasser. Bacteria live
under constant threat of attack by a virus, so they have developed incredibly effective defenses
against those threats.
The problem, Johnston explained, is that when scientists want to place human-made
DNA into bacteria, they confront the exact same defense systems that protect bacteria against a
virus.To get past this barrier, scientists add specific modifications to disguise the human-made
DNA and trick the bacterium into thinking the intruder is a part of its own DNA. This approach
sometimes works but can take considerable time and resources. Johnston's strategy is different.
Instead of adding a disguise to the human-made DNA, he removes a specific component of its
genetic sequence called a motif. The bacterial defense system needs this motif to be present to
recognize foreign DNA and mount an effective counter-attack. By removing the motif, the
human-made DNA becomes essentially invisible to the bacterium's defense system.
"Imagine a bacterium like an enemy submarine in a dry-dock, and a human-made
genetic tool as your soldier that needs to get inside the submarine to carry out a specific task.
The current approaches would be like disguising the spy as an enemy soldier, having them walk
up to each gate, allowing the guards to check their credentials, and if all goes well, they're in,"
Johnston said. "Our approach is to make that soldier invisible and have them sneak straight
through the gates, evading the guards entirely. "This new method requires less time and fewer
resources than current techniques. In the study, Johnston used Staphylococcus aureus bacteria
as a model, but the underlying strategy he developed can be used to sneak past these major
defense systems that exist in 80 to 90 percent of bacteria that are known today.
This new genetic engineering tool opens up the possibilities for research on bacteria that
haven't been well studied before. Since scientists have a limited amount of time and resources,
they tend to work with bacteria that have already been broken into, Johnston explained. With
this new tool, a major barrier to breaking into bacteria DNA has been solved, and researchers
can use the method to engineer more clinically relevant bacteria. "Bacteria are the drivers of our
planet," said Dr. Gary Borisy, a Senior Investigator at the Forsyth Institute and co-author of the
paper. "The capacity to engineer bacteria has profound implications for medicine, for agriculture,
for the chemical industry, and for the environment."
This research was facilitated by an 'NIH Director's Transformative Research Award'
(R01OD024734) granted to the research team in 2017 through the National Institute of Dental
and Craniofacial Research (NIDCR). (science daily.com)

The next lesson will discuss about recombinant DNA technology and its applications.

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Lesson

2 Recombinant DNA Technology

Recombinant DNA technology has been widely used in improving crop varieties. With
the use of this technique, several transgenic or genetically modified organisms(GMO) have
been produced. This part of the module will help you discuss the applications of Recombinant
DNA technology.

What is It

Although recombinant DNA technology first emerged in the 1960s and 1970s, the basic
principle of recombination had been discovered many years earlier. Indeed, in 1928, Frederick
Griffith, an English medical officer studying the bacteria responsible for a pneumonia epidemic
in London, first demonstrated what he termed "genetic transformation"; here, living cells took up
genetic material released by other cells and became phenotypically "transformed" by the new
genetic information. More than a decade later, Oswald Avery repeated Griffith's work and
isolated the transforming molecule, which turned out to be DNA. These experiments showed
that DNA can be transferred from one cell to another in the laboratory, thus changing the actual
genetic phenotype of an organism.

Prior to these classic experiments, the idea that the genetic material was a specific
chemical that could be modified and transferred into cells was certainly controversial. But before
the explosion in recombinant DNA could begin, scientists would have to learn not only how to
transfer DNA, but also how to isolate and modify individual genes.

Key Developments in Recombinant DNA Technology

Four key developments helped lead to construction of the first recombinant DNA
organism (Pray, 2008). The first two developments revolved around how scientists learned to
cut and paste pieces of DNA from different genomes using enzymes. The latter two events
involved the development of techniques used to transfer foreign DNA into new host cells.

1. Discovering the Cut-and-Paste Enzymes

The first major step forward in the ability

to chemically modify genes occurred when
American biologist Martin Gellert and his
colleagues from the National Institutes of Health
purified and characterized
an enzyme in Escherichia coli responsible for
the actual joining, or recombining, of separate
pieces of DNA (Zimmerman et al., 1967). They
called their find "DNA-joining enzyme," and this
enzyme is now known as DNA ligase. All living
cells use some version of DNA ligase to "glue
together" short strands of DNA
during replication. Using E. coli extract, the
researchers next showed that only in the Figure 3. Recombinant Vector Construction
presence of ligase was it possible to repair by Homologous Recombination in vivo
single-stranded breaks in λ phage DNA.
https://www.elledgelab.med.harvard.edu
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(Discovered in 1950 by American microbiologist Esther Lederberg, λ phage is a virus particle
that infects E. coli.) More specifically, they showed that the enzyme was able to form a 3'-5'-
phosphodiester bond between the 5'-phosphate end of the last nucleotide on one DNA fragment
and the 3'-OH end of the last nucleotide on an adjacent fragment. The identification of DNA
ligase was the first of several key steps that would eventually empower scientists to attempt
their own recombination experiments—experiments that involved not just recombining the DNA
of a single individual, but recombining DNA from different individuals, including different species.

A second major step forward in gene

modification was the discovery of restriction
enzymes, which cleave DNA at specific
sequences. These enzymes were discovered at
approximately the same time as the first DNA
ligases by Swiss biologist Werner Arber and his
colleagues while they were investigating a
phenomenon called host-controlled restriction of
bacteriophages. Bacteriophages are viruses that
invade and often destroy their bacterial host cells;
host-controlled restriction refers to the defense
mechanisms that bacterial cells have evolved to
deal with these invading viruses. Arber's team
discovered that one such mechanism
Figure 4. Restriction Enzyme is enzymatic activity provided by the host cell.
https://www.wikidoc.org
The team named the responsible enzymes
"restriction enzymes" because of the way
they restrict the growth of bacteriophages.
These scientists were also the first to
demonstrate that restriction enzymes
damage invading bacteriophages by
cleaving the phage DNA at very specific
nucleotide sequences (now known as
restriction sites). The identification and
characterization of restriction enzymes
gave biologists the means to cut specific
pieces of DNA required (or desired) for Figure 5. A Bacteriophage
subsequent recombination. https://www.xvivo.net

2. Inserting Foreign DNA into a New Host Cell

Although Griffith and Avery had had demonstrated the ability to transfer foreign genetic
material into cells decades earlier, this "transformation" was very inefficient, and it involved
"natural" rather than manipulated DNA. Only in the 1970s did scientists begin to use vectors to
efficiently transfer genes into bacterial cells. The first such vectors were plasmids, or small DNA
molecules that live naturally inside bacterial cells and replicate separately from a bacterium's
chromosomal DNA.

Plasmids' utility as a DNA shuttle, or vector, was discovered by Stanford University

biochemist Stanley Cohen. Scientists had already established that some bacteria had what
were known as antibiotic resistance factors, or R factor-plasmids that replicated independently
inside the bacterial cell. But scientists knew little about how the different R factor genes
functioned. Cohen thought that if there were an experimental system for transforming host
bacterial cells with these R-factor DNA molecules, he and other researchers might be able to
better understand R-factor biology and figure out exactly what it was about these plasmids that
made bacteria antibiotic-resistant. He and his colleagues developed that system by
demonstrating that calcium chloride-treated E. coli can be genetically transformed into antibiotic-
resistant cells by the addition of purified plasmid DNA (in this case, purified R-factor DNA) to the
bacteria during transformation (Cohen et al., 1972).

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3. Recombinant Plasmids in Bacteria

The following year, Stanley Cohen and his

colleagues were also the first to construct a novel
plasmid DNA from two separate plasmid species
which, when introduced into E. coli, possessed all
the nucleotide base sequences and functions of
both parent plasmids. Cohen's team
used restriction endonuclease enzymes to cleave
the double-stranded DNA molecules of the two
parent plasmids. The team next used DNA ligase
to rejoin, or recombine, the DNA fragments from
the two different plasmids (Figure 6). Finally, they
introduced the newly recombined plasmid DNA
into E. coli. The researchers were able to join two
DNA fragments from completely different
plasmids because, as they explained, "the
nucleotide sequences cleaved are unique and
self-complementary so that DNA fragments
produced by one of these enzymes can associate
by hydrogen-bonding with other fragments
produced by the same enzyme" (Cohen et al.,
1973).

The same could be said of any DNA—not

just plasmids—from two different species. This
universality—the capacity to mix and match DNA
from different species, because DNA has the
same structure and function in all species and
Figure 6. Recombinant Plasmid because restriction and ligase enzymes cut and
https://www.eng.utoledo.edu paste the same ways in different genomes—
makes recombinant DNA biology possible.

Today, the E. coli λ bacteriophage is one of the most widely used vectors used to carry
recombinant DNA into bacterial cells. This virus makes an excellent vector because about one-
third of its genome is considered nonessential, meaning that it can be removed and replaced by
foreign DNA (i.e., the human DNA being inserted as illustrated in Figure 5).

4. Vectors Used in Mammalian Cells

A fourth major step forward in the field of
recombinant DNA technology was the discovery of a
vector for efficiently introducing genes into mammalian
cells. Specifically, researchers learned that recombinant
DNA could be introduced into the SV40 virus, a pathogen
that infects both monkeys and humans. Indeed, in 1972,
Stanford University researcher Paul Berg and his
colleagues integrated segments of λ phage DNA, as well
as a segment of E. coli DNA containing the
galactose operon, into the SV40 genome. (The E.
coli galactose operon is a cluster of genes that plays a
role in galactose sugar metabolism.) The significance of
their achievement was its demonstration that recombinant
DNA technologies could be applied to essentially any
DNA sequences, no matter how distantly related their
species of origin. In their words, these researchers
"developed biochemical techniques that are generally
applicable for joining covalently any two DNA molecules" Figure 7. Introduction of Genes into a
Mammalian Cell using a Vector
https://www.bioxys.com
8
(Jackson et al., 1972). While the scientists didn't actually introduce foreign DNA into a
mammalian cell in this experiment, they provided (proved) the means to do so.

Recombinant DNA Technology Creates Recombinant Animals

The first actual recombinant animal cells weren't developed until about a decade after
the research conducted by Berg's team, and most of the early studies involved mouse cells. In
1981, for example, Franklin Costantini and Elizabeth Lacy of the University of Oxford introduced
rabbit DNA fragments containing the adult beta globin gene into murine (mouse) germ-line cells
(Costantini & Lacy, 1981). (The beta globins are a family of polypeptides that serve as the
subunits of hemoglobin molecules.) Another group of scientists had demonstrated that foreign
genes could be successfully integrated into murine somatic cells, but this was the first
demonstration of their integration into germ cells. In other words, Costantini and Lacy were the
first to engineer an entire recombinant animal (albeit with relatively low efficiency).

Interestingly, not long after the publication of his team's 1972 study, Paul Berg led a
voluntary moratorium in the scientific community against certain types of recombinant DNA
research. Clearly, scientists have always been aware that the ability to manipulate the genome
and mix and match genes from different organisms, even different species, raises immediate
and serious questions about the potential hazards and risks of doing so—implications still being
debated today.

Since these early studies, scientists have used recombinant DNA technologies to create
many different types of recombinant animals, both for scientific study and for the profitable
manufacturing of human proteins. For instance, mice, goats, and cows have all been
engineered to create medically valuable proteins in their milk; moreover, hormones that were
once isolated only in small amounts from human
cadavers can now be mass-produced by genetically
engineered cells. GloFish (Figure 8) are a type of
transgenic zebrafish (Danio rerio) that have been
modified through the insertion of a green
fluorescent protein (gfp) gene. Not all GloFish are
green, however. Rather, there are several gfp gene
constructs, each encoding a different
colored phenotype, from fluorescent yellow to
fluorescent red. In fact, the
entire biotechnology industry is based upon the
ability to add new genes to cells, plants, and
animals As scientists discover important new
proteins and genes, these technologies will continue
to form the foundation of future generations of
discoveries and medical advances. Figure 8. Glofish
https://www.sawse.com
Recombinant DNA Technology in Plants
Typically, the vector used to insert genes into plant cells is the Ti plasmid, from the soil
bacterium Agrobacterium tumefaciens. This bacterium normally causes the crown gall disease
in plants by using its Ti, or tumor-inducing plasmid to insert into plant cells. The plasmid
incorporates a portion of its DNA into the
DNA of the plant cells. Genetically modified
pants are produced by integrating a gene of
interest into the Ti plasmid before inserting
the plasmid into the plant cells. These now
possess genes that would confer traits such
as resistance to certain bacterial or fungal
pests. For example, genetically engineered
corn, also called ‘Bt corn’ expresses a gene
from the soil-dwelling bacterium Bacillus
thuringiensis, making them resistant to the
corn borer disease. Other examples include
Figure 9. An organic and a genetically the ‘golden rice’ which is a transgenic variety
modified tomato. of rice that is engineered to produce beta-
https://www.wakingtimes.com

9
carotene and prevent vitamin-A deficiency; rice and potato have been modified to produce
harmless proteins derived from cholera to serve as a natural vaccine and; soybean has been
engineered to have resistance against herbicides thus the entire field can be exposed to
herbicides in order to destroy the weeds without harming the crops.

In agriculture development of genetically modified crops with a purpose to improve both

yield and resistance to plant pests or herbicides seems to have gained a degree of public
acceptance and is already practised in a commercial context in several countries.4 The
genetically modified tomato CGN-89564-2 was the first commercially grown, genetically
engineered crop product to be granted a licence for human consumption. This was developed in
1994 to express the trait of delayed softening of tomato flesh as a practical means to minimize
post-harvest crop losses.5 Ironically given its brand name of ‘Flavr Savr’, this failed in the
marketplace due not to public apprehension over eating a genetically altered food per se but to
an apparent lack of taste. Nevertheless, the introduction of a genetically modified fruit paved the
way for use of GMOs in food and today genetic modification is widespread.

There are many different traits that can be introduced to organisms to change their properties.
The following table shows examples of modified traits using cloned genes and their applications:

MODIFIED GENE MODIFICATION RECIPIENT APPLICATION (FIELD)

TRAIT ORGANISM
Insulin Insertion of Human (Medicine) Production of Human Insulin in
Production Insulin Gene Bacteria Bacteria

(Agriculture) Production of corn plants with

Pest Insertion of Bt-toxin Corn/ Maize increased resistance to corn boxer
Resistance gene

(Agriculture) Production of plants with

Disruption of a gene for Tomato plant fruits that have delayed ripening. These
Delayed a ripening enzyme fruits will survive longer transport time,
Ripening (e.g.polygalacturonase) allowing their delivery to further locations
(i.e.export deliveries)
(Industry) Enhance large scale production
of chymosin. This enzyme serves as a
substitute for rennetin the coagulant of
Chymosin Insertion of a gene for Bacteria milk.The large scale production of this
Production chymosin enzyme in bacteria Rennethastobe
harvested from calves provides an
abundant supply of this important
component for the cheese production
industry

A general outline of recombinant DNA may be given as follows:

1. Cutting or cleavage of DNA by restriction enzymes(REs).

2. Selection of an appropriate vector or vehicle which would propagate the recombinant

DNA(eg.circular plasmid in bacteria with a foreign gene of interest).

3. ligation(join together) of the gene of interest(eg.from animal) with the vector(cut bacterial
plasmid).

4. Transfer of the recombinant plasmid into a host cell(that would carry out replication to
make Huge copies of the recombined plasmid).

5. Selection process to screen which cells actually contain the gene of interest.

6. Sequencing of the gene to find out the primary structure of the protein.

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Ways in which these plasmids may be introduced into host organisms:

 Biolistics. In this technique, a “gene gun” is used to fire DNA-coated pellets on

plant tissues. Cells that survive the bombardment, and are able to take up the expression
plasmid coated pellets and acquire the ability to express the designed protein.

 Plasmid insertion by Heat Shock Treatment. Heat Shock Treatment is a

process used to transfer plasmid DNA into bacteria. The target cells are pre-treated before
the procedure to increase the pore sizes of their plasma membranes. This pre-treatment
(usually with CaCl2) is said to make the cells “competent” for accepting the plasmid DNA.
After the cells are made competent, they are incubated with the desired plasmid at about
4°C for about 30min. The plasmids concentrate near the cells during this time. Afterwards,
a “HeatShock” is done on the plasmid-cell solution by incubating it at 42°C for 1 minute then
back to 4°C for 2 minutes. The rapid rise and drop of temperature is believed to increase
and decrease the pore sizes in the membrane. The plasmid DNA near the membrane
surface are taken into the cells by this process. The cells that took up the plasmids acquire
new traits and are said to be “transformed”.

 Electroporation. This technique follows a similar methodology as Heat Shock

Treatment, but, the expansion of the membrane pores is done through an electric “shock”.
This method is commonly used for insertion of genes into mammalian cells.

Other methods include:

 Selection of plasmid DNA containing cells

 Selection of transformed cells with the desired gene

 PCR detection of plasmid DNA

Ethical Considerations

In evaluating eikaryotic organisms as suitable for genetic engineering, there are ethical
issues to be considered, such as the possibility of GMOs released into the environment as bio-
controlling agents becoming pathogenic to non-harmful organisms. Notably, this occured for the
Entomopathogenic hyphomycetous fungi in Lagenidium, Coelomomyces and Culicinomyces
used to kill Anoplheles and Aedes mosquito larvae as supposedly environmentally friendly
means to combat the major vector-borne diseases malaria and dengue.

In choosing to exploit r-DNA technology for developing novel GMOs public education
should be an important consideration. A high level of acceptance is required in order to attain
societal trust in and use of a given product and thereby to achieve its economic success. Ethical
concerns should be addressed by rolling out effectively communicated information campaigns
and by designing strategies for stronger community engagement.

A. Agriculture

The introduction of pest-resistant brinjal (also known as eggplant or aubergine) was met
with criticism in some countries, in contrast to the concurrent popularity of pest-resistant cotton.
Both attempts at implementation followed incorporation of the identical crystal protein gene
(Cry1Ac) from the soil bacterium Bacillus thuringiensis (Bt) into the genome of the host plant
expression of which synthesizes so-called Bt toxins that confer resistance to predation by
lepidopteran insects. However, of the two uses as a food and as clothing the one which caused
anxiety among the general public involved human consumption. The benefits to humans of
using Bt toxin should be stressed in an attempt to overcome the initial unpopularity of
consuming Bt-brinjals in developing countries such as India, Bangladesh and Philippines. This
would reduce public scepticism founded on the misperception that eating a plant product
containing a ‘toxin’ is in fact toxic to humans irrespective of its unrelated target and benign
mechanism of action.

11
B. Medicine

Drug delivery systems in medicine that are based on bacterial or viral hosts could prove
hazardous if either the organism is genetically unstable and converts to a pathogenic type or if
purification is incomplete. In an analogous proof of concept from the agricultural sphere, use of
the soil bacterium Agrobacterium tumefaciens as a vehicle for gene transfer is very effective
and has become widely adopted despite its tumorigenicity, causing crown gall disease of
dicotyledonous plants. Genetic reversion is also a major concern regarding the experimental
technique of gene therapy to treat or prevent otherwise incurable genetic disorders and
acquired diseases, research into which was slowed in the early 2000s due to cases of viral
vector instability. Consequently, identification of a preferred system to safely and efficiently
deliver an altered gene of choice has become a priority as the technology advances from
development and laboratory research to clinical translational trials.

C. Bioremediation

Pseudomonas putida and Nitrosomonas europaea are the organisms which are typically
utilized in bioremediation. The objective is to isolate the original genes located in these bacteria
that promote bioremediation, then modify and incorporate them into a suitable host to be used
as a bioremediation agent usually E. coli. This may, however, impact normal ecosystems as
well; for example, bacteria that have an improved ability to digest petroleum could, if exposed,
cause destruction of important petroleum products. Hence, stringent monitoring of in situ
bioremediation is essential. In producing genetically modified bacteria the simplest way of
screening is to incorporate a marker gene, which, typically is one that confers antibiotic
resistance. This achieves the purposeful generation of antibiotic-resistant organisms which, if
mishandled, could become problematic under natural conditions.

D. Biotechnology

An appreciable biotechnological success and novel commercial application is the

production of genetically modified fluorescent zebrafish, Danio rerio, and similar species using
genes encoding glowing characteristics. This is marketed under the GloFish® patent in the US
where fish coloured bright red, green, orange-yellow, blue and purple are sold as pets to be kept
in the controlled environment of an indoor aquarium. In the event of release, inadvertent or
deliberate, into the environment the survival capacity of these constantly fluorescent fish is
markedly reduced due to increased vulnerability to predation compared to wild type fish; thus,
the risk of sustained ecological impact is considered to be marginal. However, in-depth research
to confirm or refute this notion is currently not possible because of insufficient understanding
and a lack of technology to study the nexus of evolutionary biology and ecology with specific
reference to the introduction of a novel species into, and its subsequent migration from, an
ecosystem.

Key points:
 Recombinant DNA technology is not limited to plants. Recombinant bacterial cells with
human genes can be used in order to produce human proteins.

 Genetic engineering and Recombinant DNA technology has always been a topic of
controversy as the balance it aims to reach between the benefits accrued to humans and
attendant ethical considerations is open to debate. In each of the diverse fields of
agriculture, medicine, bioremediation and biotechnology concerns vary in a discipline-
specific manner. However, the principal source of apprehension often involves the
ecological impact, real or perceived, of the use of recombinant DNA technology, in
particular the release of genetically modified organisms into the environment.

 Matters of contention surround such fundamental aspects as the creation of organisms

containing an altered genome and the inheritance of modified genes by the offspring of
such animals or plants. These should be addressed in greater detail and with considerable
circumspection since there may be robust counter arguments regarding each issue. In

12
 agriculture, for example, these include the possibility of elimination of wild type plant
cultivars in the absence of insect pest resistance, insects developing resistance, elimination
of organisms which consume modified plant material, and existing non-target secondary
pests becoming primary pests. The ethical right of humans to intervene with nature through
‘artificial selection’ by altering genomes has been questioned, eliciting a critical outcry in
Europe during the introduction of GMOs. Hence, it is important to determine the possibility
of modified genes being passed to future generations as well as their effects on the
ecosystem.

What’s More

Learning the concepts about Genetic Engineering and Recombinant DNA Technology
opens up a whole new perspective. Have fun in the activities below!
Activity # 1: Recombinant DNA Technology
Goal: To understand the concepts of Recombinant DNA Technology.

Objective: Students will…

 Discover new medical techniques that are being used to treat diseases using DNA.

 Complete a paper lab to explore the possibilities of the use of recombinant DNA.
Read points 1-5
1. How and why do we engineer human genes into bacterial DNA? How do we isolate and
manipulate genes in which we are interested? One method scientists commonly use is called
recombinant DNA technology. Recombinant DNA technology is the process of cutting and
recombining DNA fragments. Usually human DNA containing genes for a particular protein are
used, recombined with bacterial DNA and then inserted into a bacterial cell (transformation).
Recombinant DNA technology coupled with the knowledge of transformation opens many doors
in genetic engineering. If scientists can alter DNA, they can then insert desired genes into
another organism. They can alter the genes of bacteria to cause them to produce a desired
human protein product.
2. Once a gene is sequenced, it can be used in recombinant DNA techniques. Sequencing is a
technique used to determine the order of genetic information in DNA. For example the
sequence of a gene might begin as C A T A T G. One of the first genes sequenced was the
gene that codes for insulin, a hormone that regulates blood sugar. Another gene of interest is
the gene p53. p53 (also known as TP53) is a tumor suppressing gene. It produces a protein
that will regulate the cell cycle by inhibiting cells from growing and dividing too quickly. This
protein is contained in the nucleus of body cells and will bind to the DNA determining whether
the DNA will be repaired or whether the cell will undergo apoptosis (programmed cell death) if
the DNA becomes damaged by mutagens such as toxic chemicals, UV light, or viruses. This
process prevents the development of tumors by stopping cells with damaged DNA from
undergoing mitosis and passing down this damaged DNA to daughter cells. If it is determined
that the DNA can be repaired p53 will activate other genes to fix the genetic damage. Due to
the activity of p53 of regulating cell division, this gene has been called the “guardian of the
genome” ,“the guardian angel gene” or the “master watchman”.
3. A plasmid is a circular, double stranded piece of DNA that occurs naturally in bacteria and
can be used as an important tool in genetic engineering. A human gene can be inserted into a
plasmid (this is used as a vector to transfer the gene into a bacterial cell), and then this DNA is
absorbed by a host cell such as E.coli . This bacterial cell becomes transformed with the
recombinant DNA, and the gene is expressed. In a laboratory this transgenic bacteria is cloned
and the plasmid would then be replicated, transcribed and translated into a protein in the host

13
cell. Many drugs are now manufactured this way. Scientists insert a gene coding for the desired
protein into a bacteria and the desired trait is expressed.
4. The process of transformation allows bacteria to take in foreign DNA. This occurs in nature
but when bacteria are transformed in the lab a plasmid containing a gene for antibiotic
resistance is used so the transgenic E.coli containing the recombinant DNA can be located.
5. In this activity you will be a molecular biologist! You will use a paper model to simulate
recombinant DNA technology by identifying the p53 gene on chromosome 17, cutting it out and
putting it into a plasmid. Using materials provided for your simulation, follow the steps below to
isolate the gene and put it in a plasmid. You will simulate standard techniques used in
recombinant DNA technology in this activity.
Materials – for each team of 2 students:
Plasmid handout
Tape
P53 Gene handout
Highlighter marker
Restriction enzymes handout
Scissors

Directions:
Part 1

 Collect the materials you need:

Plasmid handout
P53 Gene handout
Restriction enzymes handout
Scissors
Tape
Highlighter marker

 Create your own plasmid. Many plasmids that are used in research laboratories are
made synthetically (by human intervention). Scientists build plasmids according to how
they use them.
 To create your own plasmid follow the steps below:
1. Cut out the double stranded DNA sequence from the plasmid handout. Be
sure to cut along the dotted lines.
2. Tape the sections together end to end.
Hint: You may tape the plasmid strips together in any order.
3. Tape the ends of the entire strip together so that the plasmid is circular.
Make sure the circle is such that you can see the base pairs on the
outside.
 Now, create your own chromosome 17 by cutting out the double stranded genomic DNA
sequence from the p53 gene handout. Cut along the dotted line and tape the sections
together end to end in numerical order.
Hint: Be sure to tape the strips representing the chromosome in order.
Chromosomes are not built according to scientists needs. Scientists discover and study
them as they naturally exist.

14
Questions for thought:
1. What are the differences between a plasmid and a chromosome?
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
2. What would a scientist need to do before he or she could remove a gene from a chromosome?
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________

 Now that you have a plasmid and a chromosome, you are going to use recombinant
DNA technology to move genes. Read the following paragraph.

Restriction enzymes are another important tool that scientists use. Essentially, they work
like scissors that cut at specific locations along a DNA strand. There are thousands of restriction
enzymes that occur naturally in bacteria. Most likely, their function in bacteria is to cut up foreign
DNA. Scientists use restriction enzymes as a tool in molecular biology. Restriction enzymes
work by cutting DNA at specific locations along the DNA sequence. Each enzyme cuts at a
specific DNA sequence called a restriction site.
Your scissors will be used as restriction enzymes in this activity. On the restriction
enzymes handout, several restriction enzymes are listed next to the DNA sequence at which
they cut.

 Study the DNA sequences at which the restriction enzymes cut on the restriction
enzymes handout. Discuss your understanding of the restriction site with your partner.
 On chromosome 17, locate the restriction sites described in the restriction enzyme
handout. Label all of the places along the chromosome where a restriction enzyme
would be cut. Be sure to label each site with the name of the restriction enzyme and
draw a line indicating where the enzyme will cut. Note: not every enzyme will cut along
these sections of DNA.
 Now think about which restriction enzyme(s) you can use to cut out the p53 gene.
Highlight the sites where you can cut the restriction enzymes you would use. Do not cut
out the gene yet.
Questions for thought:

3. Which restriction enzyme(s) would you use to cut out the p53 gene? Why?
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
4. What other information might you need before making your final choice?
Hint: Your goal is to put the p53 gene into the plasmid.
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________

 When you cut out the p53 gene, you will need a place to put it for processing. We can
use Plasmid DNA for this purpose. In fact, plasmids can serve as vectors. Vectors are
used to carry a gene to an organism. The gene within the plasmid can then be replicated,

15
 transcribed, and translated all within a host organism, such as the bacteria E.coli.
Plasmids use the machinery of the host bacteria to accomplish this feat. Locate
restriction sites on the plasmid DNA using the restriction enzyme handout as a guide.
Label these sites with the name of the restriction enzyme and draw a line indicating
where the enzyme will cut.
 Compare the restriction sites you found on both the chromosome and the plasmid.
Knowing that the p53 gene needs to be placed into the plasmid, identify which restriction
enzyme(s) you should use to cut out the p53 gene and to cut the plasmid DNA.
Hint: The plasmid is used as a vector (a device to carry the gene). You do not
need to remove DNA form the plasmid. You will only need to open up the plasmid to
insert the p53 gene. You might accomplish this by using one enzyme.
 Once you have decided upon which restriction enzyme to use, check with your teacher
before you actually start cutting. Using the restriction enzymes handout as a guide, use
your scissors as a restriction enzyme to cut the DNA sequence at the sites you have
identified.
 Remove the p53 gene from the chromosome 17. Isolate the gene by removing the rest
of the DNA (throw it away).
 On your plasmid, cut the DNA sequence at the site(s) you have identified.

Questions for thought:

5. What happens to the plasmid when you cut it? How many pieces of DNA do you have? What
happens to chromosome 17? How many pieces do you get?
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
6. Compare the ends of the plasmid DNA with the ends of the isolated p53 gene. What do you
notice?
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
Read the following paragraph, which describes the different ways restriction enzymes
work.

When studying the restriction sites, did you notice differences in how the enzymes cut
DNA? For example, Eco RI cuts between the G and A. This leaves what is called a “sticky end”
on both ends of the DNA. Sometimes the cut leave a “blunt end”, like the Hpa I restriction
enzyme. The illustration below of (a) and (b) shows double stranded DNA cut with a restriction
enzyme. The top lines represent one strand and the bottom line represents the complementary
strand. The spaces represent where the enzymes have cut. (a) shows DNA cut with an enzyme
leaving sticky ends and (b) shows DNA cut with an enzyme leaving blunt ends.
___________ _____________ ____________ ___________
_______________ _______ ____________ ___________
(a) (b)

 It is now time to put the p53 gene in the plasmid. Another enzyme, called ligase, assists
in the formation of bonds between adjacent, matching DNA ends. Your tape will play the
role of the ligase. Insert the p53 gene in the plasmid DNA in the appropriate place. Tape
the ends together. Does it fit?

16
Questions for thought:
7. What is the role of the plasmid?
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
8. What is the role of the gene?
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
9. Do you think the direction of the gene might be important? Why or why not?
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
Discuss the following questions:
10. Was every group successful in putting the p53 gene into a plasmid? Why or why not?
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
11.Why is the location of the restriction site important? Which sites work and which wouldn’t?
Why?
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
Activity # 2: Recombinant DNA Technology
Additional Questions for Research or Thought: Students Sheet
Name:_______________________________________Class Period:________________
1. How does a molecular biologist manipulate the human gene to take care of the
problem with introns?
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
2. What other combinations of DNA could result after treating the cut plasmid DNA and
p53 gene with ligase?
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
3. Will bacteria transform with all of the above possible combinations of DNA?
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________
____________________________________________________________________________

17
 What I Have Learned

Congratulations! You passed a lot of engaging activities. This time, you need to synthesize what

you have learned by doing the task below.

Instruction: Analyze the figure to do the task below.

Figure 10. Sheep cloning

www.slideshare.net

1. In the cloning shown in Figure 9, which sheep is the source of the nucleus in the fused cell?

2. In Figure 9, why was the nucleus removed from the egg cell?

3. Which animal in Figure 9 is a clone?

4. In the cloning shown in Figure 9, which sheep provided an egg cell?

5. Which two animals in Figure 9 are genetically identical?

18
 What I Can Do

DNA Technology
1. Name two situations when DNA fingerprints are useful.
_________________________________ and _______________________________
2. How does the DNA migrate from one end of the gel to the other?
3. What cuts up the DNA into tiny fragments? _____________________________________
Use the gels below to answer the following questions. felon S1 S2 S3
dad dad
baby mom
1 2 5. Lt. Russ is investigating a
murder scene. The felon was
scratched by his victim & some
of his skin cells were found
under the victim’s fingernails.
A DNA test was performed.
Which of the suspects is the
murderer?

felon S1 S2 S3
6. Suzy was assaulted in
4. Mrs. Smith has a baby an alley. The police
named Jessica. She collected a sample of
believes one of two men cells that was left at the
can be the father of her crime scene and now
child. A paternity test is have 3 suspects in
done and the results are custody. Which of the
shown above. Which of suspects assaulted Suzy?
the 2 men are baby
Jessica’s father?
child child child child child child
dad mom
1 2 3 4 5 6

7. Mr. & Mrs. Jones just gave birth to

fraternal twins- Bob and Jane.
Unfortunately, the nurse has confused the
Jones twins with 4 other babies. The doctors
took samples of DNA from each of the
babies and Mr. & Mrs. Jones. Which of the
6 children are Mr. & Mrs. Jones twins?
mom daughter son 1 son 2

8. The millionaire, Mr. Big, has just died. He has left behind a wife, daughter and
a large inheritance. The news of his death has brought forth 2 men who claim to
be the Yes
long you didofit!Mr.
lost son You are almost
& Mrs. at the
Big. Before Mr.final task.
& Mrs. BigGet
wereready and
married be able to do it right.
they
Remember
had a child that
takenlearning is fun!
from them. They had tried to find him but had no luck in
locating him. A DNA sample was taken from Mrs. Big, the Big daughter and the
two men who claim to be the long lost son. Which, if any, of the men are telling
the truth?
19
 Assessment

Directions: Read each statement and choose the letter of the correct answer. Write your
answer on your answer sheets.

1. Which of the following enzymes in bacteria are responsible for restricting the growth of viruses?
A. Gyrase C. topoisomerase
B. protease D. restriction endonuclease
2. What carries a gene from one organism into a bacteria cell?
A. a plasmid C. an electrophoresis gel
B. a restriction enzyme D. polymerase chain reaction
3. Which enzyme is used to join together two different types of DNA molecules?
A. ligase C. exonuclease
B. protease D. endonuclease

4. Which of the following refers to the process of making changes in the DNA code of a living
organism?
A. inbreeding C.selective breeding
B. hybridization D.genetic engineering
5. What is recombinant DNA?
A. DNA that has been sequenced
B. DNA that causes genetic disorders
C. Adding DNA from one organism into the DNA of another
D. DNA which has been changed by natural selection
6. What type of insulin is produced by recombinant DNA technology?
A. A combination of E. coli and human insulin
B. Engineered to be more effective than human insulin
C. Identical to human insulin produced in the pancreas
D. Cheaper but less effective than pig insulin for treating diabetes
7. What is the ultimate source of genetic variation?
A. radiation C. inbreeding
B. mutations D. hybridization
8. It refers to the entire collection of genes within human cells is referred to as the?
A. pedigree C. gene map
B. karyotype D. human genome
9. In the reproductive cloning of an animal, the genome of the cloned individual comes from?
A. an egg cell C. a sperm cell
B. A body cell D. any gamete cell

20
10. What is a genetically modified organism?
A. a hybrid organism
B. a plant with certain genes removed
C. an organism with an artificially altered genome
D. any agricultural organism produced by breeding or biotechnology

Additional Activities

Now that you’ve learned about genetic engineering and recombinant DNA technology,
this part of the module will enrich your knowledge and learning bank.
Name: ________________________ Class: ___________________ Date: __________

Essay
1. In what ways has selective breeding been useful to humans today and in the past?

21
Answer Key

22
23
References

Johnston, Christopher D., Sean L. Cotton, Susan R. Rittling, Jacqueline R. Starr, Gary G. Borisy,
Floyd E. Dewhirst, Katherine P. Lemon. Systematic evasion of the restriction-modification
barrier in bacteria. Proceedings of the National Academy of Sciences, 2019. Retrieved from:
https://www.sciencedaily.com/releases/2019/05/190528140106.htm

Pray, Leslie. Recombinant DNA Technology and Transgenic Animals. Nature Education.2008.
Retrieved from: https://nature.com/scitable/recombinat-dna-technology-and-transgenic-
animals.34513

Robinson, Taylor AW., Rajakaruna SS. Application of Recombinant DNA

Technology(genetically modified organisms) to the Advancement of Agriculture, Medicine,
Bioremediation and Biotechnology Industries. Journal of Applied Biotechnology and Engineering.
Volume 1 Issue 3 eISSN: 2572-8466. 2016

Images

Figure 1. Plasmid Cloning.Free Royalty. https://www.davidson.bio.edu

Figure 2. Sheep Cloning.Free Royalty. https://www.slideshare.net
Figure 3. Recombinat Vector.Free Royalty. https://www.elledgelab.med.harvard.edu
Figure 4. Restriction Enzyme.Free Royalty. https://www.wikidoc.org
Figure 5. Bacteriophage.Free Royalty. https://www.xvivo.net
Figure 6. Recombinant Plasmid.Free Royalty. https://www.eng.utoledo.edu
Figure 7. Mammalian Cell.Free Royalty. https://www.bioxys.com
Figure 8. Glofish.Free Royalty. https://www.sawse.com
Figure 9. Tomatoes.Free Royalty. https://www.wakingtimes.com
Figure 10. Sheep Cloning.Free Royalty. https://www.slideshare.net
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