12

GENERAL BIOLOGY 2
Guided Learning Activity Kit
Recombinant DNA (rDNA)
Quarter 3 - Week 1

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General Biology 2 – Grade 12
Guided Learning Activity Kit
Recombinant DNA (rDNA)
Quarter 3 - Week 1

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Guided Learning Activity Kit Development Team

Writer: Argel Joseph C. Mayo
Editor: Pamie F. Malana
Reviewer: Pamie F. Malana
Illustrator: Roberto L. Pascua Jr.
Management Team: Leonardo D. Zapanta EdD, CESO V
Michelle Ablian-Mejica EdD
Manolito B. Basilio EdD
Evelyn D. Tarrayo EdD
Garry M. Achacoso
Rachelle C. Diviva
Chester Allan P. Matienzo

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 RECOMBINANT DNA (rDNA)

Introduction

Genetic engineering is the method of modifying an organism's genetic

structure by using recombinant DNA (rDNA) technology. Traditionally, by
regulating breeding and choosing offspring with desirable characteristics,
humans have implicitly manipulated genomes (National Human Genome
Research Institute).
In Recombinant DNA technology, DNA molecules are inserted into the
host organism from two separate organisms to create novel genetic
combinations that are important to science such as medicine and agriculture.
Since the gene is the focus of this technology, laboratory geneticists' central
aim is to separate, classify, and modify genes (Britannica 2021).
This learning activity kit will guide you to explore the concepts of
Recombinant DNA (rDNA) technology. It is divided into two: Lesson 1 (Genetic
Engineering); Lesson 2 (Application of DNA Technologies).

Learning Competencies

● Outline the processes involved in genetic engineering (STEM_BIO11/12-

IIIa-b-6)
● Discuss the applications of recombinant DNA (STEM_BIO11/12-IIIa-b-7)

Objectives

At the end of this guided earning activity kit, you are expected to:
1. define recombinant DNA technology and genetic engineering;
2. discuss the structure and important roles of plasmids in genetic
engineering;
3. illustrate general techniques used by genetic engineers to modify DNA;
and

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4. create a poster / educational video that shows concepts and applications
of recombinant DNA technology in the field of medicine and agriculture.

Review

Deoxyribonucleic Acid (DNA) and Ribonucleic Acid are the two main
nucleic acids. In all living species, DNA, the genetic material is present and
can be found in cells of microorganisms (ex. Bacteria) and animals, while RNA
is involved in protein synthesis.
Let us have a review of the basic structure of DNA and RNA by choosing
the right answers to complete the table below.

Structure DNA RNA

Pentose Sugar
Base Composition

Base Pairing

Number of Strands

Deoxyribose Guanine (G)

Ribose Cytosine (C)
Double strand Thymine (T)
Single strand Uracil (U)
Adenine (A)

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 Discussion

LESSON 1: GENETIC ENGINEERING

OVERVIEW OF GENETIC ENGINEERING

● Genetic engineering requires one or more genes to be specifically
manipulated. A gene from another animal is most applied to the genome of
an organism to give it a phenotype that is desired (National Human Genome
Research Institute).
● As the first researcher to create a recombinant DNA molecule, Paul Berg, a
molecular biologist, shared the Nobel Prize in Chemistry in 1980 for his
contributions to recombinant DNA technology.
● In rDNA technology, scientists use restriction enzymes from bacteria to
cut DNA molecules only in particular locations (Solomon, Berg, and Martin
2011, 389).

PLASMIDS: IMPORTANT TOOLS FOR GENETIC ENGINEERING

● A plasmid is a small, double-stranded, circular DNA molecule that is
distinct from the chromosomal DNA of a cell.
● In bacterial cells, plasmids occur normally.
● The genes carried in plasmids also give genetic benefits to bacteria, such as
antibiotic resistance.
● All of the plasmids found within the cell are copied as a bacterium undergo
cell division so that each daughter cell gets a copy of each plasmid.
● In order to use them as instruments to replicate, transfer, and modify genes,
scientists have used plasmids.
● Plasmids that are used experimentally for these purposes are called vectors
(a vector is a carrier capable of transporting the DNA fragment into a cell).
● In a plasmid vector, researchers may insert DNA fragments or genes,
producing a so-called recombinant plasmid.
● By means of a mechanism called transformation (the uptake of foreign DNA
by cells), this plasmid can be introduced into a bacterium.
● Since bacteria divide easily, they can be used as factories to copy vast
amounts of DNA fragments (Nature Education 2014).

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Figure 1: Structure of Bacterial Cell. It Contains a Single Copy of the
Plasmid.

STRUCTURE OF PLASMID DNA

● Ori / Origin of Replication
The plasmid has its own process for replication and transcription
since it has DNA sequences that allow autonomous plasmid DNA
replication.
● Antibiotic Resistance Gene
One of the peculiar features of the plasmid is the antibiotic
resistance gene. The plasmid antibiotic resistance gene helps bacteria
survive and protects them against antibiotics.
● Restriction Site
During the experiment, the restriction site is used for the
insertion of a gene of interest.
● Promoter region
Transcription of target DNA is encouraged by the promoter
region.
● Selectable Marker
The selectable marker enables bacteria with the gene of interest
to be selected. The gene or gene of interest is used to study and modify
it.

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● Gene of Interest
The particular gene being studied or manipulated in an
experiment (Genetic Education 2020).

Figure 2: The General Structure of Plasmid DNA in Recombinant DNA

Technology

STAGES OF GENETIC ENGINEERING

1. Identify the section of DNA that contains the required gene from the source
chromosome.
2. Extract the required gene.
3. Insert required gene into vector / bacterial plasmid.
4. Insert plasmid into the host cell.
5. Grow transformed cells to produce a GMO.

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What is Genetically Modified Organism (GMO)?
It is an organism (animal, plant, microorganism, or other organisms)
whose genetic makeup has been modified using genetic engineering.
It is performed in the laboratory to create a combination of genes of
animal, plant, or bacteria that do not happen in nature or traditional
crossbreeding techniques.
We will discuss some examples of GMOs in the next lesson.

LESSON 2: APPLICATIONS OF DNA TECHNOLOGIES

Recombinant DNA technology has developed a modern and unique

range of methods to resolve basic cell concerns, as well as new approaches to
problems in many fields. The development of genetically modified proteins,
crops, and animals has started to have a considerable influence on our lives
in certain areas. The most striking of these has been in medicine and
agriculture.
Let us discuss the use of DNA technology in these fields:

1. MEDICINE

A. Human Insulin Produced by Escherichia coli

- In contemporary biotechnology, the Escherichia coli bacterium
(E. coli for short) is essential. Scientists use rDNA technologies to
manufacture proteins such as insulin.
- Insulin is a hormone produced by the beta cells of the islets of
Langerhans found in the pancreas. It allows cells to absorb
glucose (sugar) from the blood. It also regulates the blood glucose
level in our body.
- Human insulin provided by E. Coli was one of the first
genetically engineered proteins to be certified for human
consumption.
- Insulin was extracted solely from other species before the
introduction of rDNA techniques to produce genetically
engineered bacteria capable of generating the human hormone.
Many diabetic patients are allergic to animal insulin because the
sequence of amino acids varies significantly from that of human
insulin.

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- The ability to develop human hormones using rDNA techniques
has contributed to important medical benefits for diabetic
patients (Solomon, Berg, and Martin 2011, 407).

Genetic Engineering Process to Produce Insulin

1. From bacteria, a small piece of circular DNA called a plasmid

is extracted.
2. By restriction enzymes (act as molecular scissors), a small
segment is then cut out of the circular plasmid.
3. The plasmid is now genetically modified after the gene for
human insulin is inserted into the gap in the plasmid.
4. The genetically modified plasmid is introduced into a new
bacteria.
5. To start making insulin, this cell then divides rapidly.
6. Genetically engineered bacteria are grown in large
fermentation vessels that provide all the nutrients they require to
generate large quantities of cells. The more the cells divide, the
more insulin is released.
7. The mixture is extracted to release the insulin after
fermentation is completed.
8. For the distribution of insulin to diabetes patients, it should
be purified and packaged into bottles and an insulin pen.
(yourgenome.org).

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Figure 3: An Illustration Showing How Genetic Modification is used to
Produce Insulin in Bacterium (E. coli)

B. Genetically Modified Human Growth Hormone (GH)

To address growth defects, especially pituitary dwarfism, it is
available to children who need it. The pituitary gland produces and
releases several hormones including growth hormone.
Long ago, human GH was obtainable from cadavers (dead bodies
used in scientific studies). But only small amounts were available.

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 Evidence also revealed that some of the human cadaver
preparations were infected with infectious agents (Solomon, Berg, and
Martin 2011, 407).

C. Development of Vaccines
rDNA technology is rapidly being used to develop vaccines that
provide humans and animals with safe and efficient protection against
infectious diseases.
Cloning a gene for a surface protein formed by the diseased agent
(pathogen) is one means of producing a recombinant vaccine.
The researcher then introduces the gene into a nondisease-
causing vector.
The vaccine induces an immune reaction to the surface-exposed
protein when administered to the human or animal host.
Consequently, the immune system targets it for degradation if
the pathogen bearing the surface protein is detected.
Vaccines for influenza A, hepatitis B, and polio are examples of
human antiviral recombinant vaccines (Solomon, Berg, and Martin
2011, 407).

2. AGRICULTURE

A. Genetically Modified Tomato

The CGN-89564-2 genetically modified tomato was the first
genetically engineered crop product to be given a human consumption
license.
This was created in 1994 as a realistic means of reducing post-
harvest crop losses to convey the characteristic of delayed softening of
tomato flesh (Bawa and Anilakumar 2013).

B. Pest Resistant Genetically Modified Crops

Pest-resistant GM crops have been genetically modified so they
are toxic to certain insects.
They are also called Bt crops because, in a bacterial species called
Bacillus thuringiensis, the added genes were originally identified.
Bacillus thuringiensis (Bt) is a soil-living species of bacteria. When
eaten, it makes proteins that are toxic to some insects. These toxins are
called Cry toxins (National Pesticide Information Center 2015).
-

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 In the Philippines, Bt corn was developed to be resistant to the
Asian corn borer (Ostrinia furnacalis). This corn borer causes wilting of
the leaves and seed failure by boring holes in the stems and pods of
plants.

C. Genetically Modified Rice (Golden Rice)

The golden rice was named because of its golden color caused by
beta-carotene (a kind of carotenoid molecule found in plants that has
orange-yellow color).
The normal rice (Oryza sativa) does not have beta-carotene on its
endosperm, that is why it usually appears off-white color.
The beta-carotene gene was inserted into a rice genome.
Once metabolized by the human body, the beta-carotene
becomes Vitamin A.
The golden rice was made to fight Vitamin A deficiency which can
cause blindness among children (The Embryo Project Encyclopedia
2013).

Activities

General Directions: Write all your answers in your answer sheet.

Guided Practice 1: Modified True or False (2 points each)

Directions: Individual Work. If the statement is correct write True, if not write
False. Then replace the word/s to make the false statement, true. Write down
your responses on separate sheets.

1. Human growth hormone provided by E. Coli was one of the first genetically
engineered proteins to be approved for human use.

2. Bacillus thuringiensis (Bt) is a soil-living species of bacteria. When eaten, it

makes proteins that are toxic to some insects. These toxins are called Cry
toxins.

3. In the past, human GH was obtainable from cadavers (dead bodies used in
scientific studies). But only small amounts were available.

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4. The CGN-89564-2 genetically modified maize was the first genetically
engineered crop product to be given a human consumption license.

5. During the experiment, the origin of replication is used for the insertion of
a gene of interest.

6. Once metabolized by the human body, Vitamin A becomes beta-carotene.

7. In the Philippines, Bt corn was developed to be resistant to the Asian corn

borer (Ostrinia furnacalis).

8. GMO is an organism (animal, plant, microorganism, or other organisms)

whose genetic makeup has been modified using genetic engineering.

9. Cloning a gene for a surface protein formed by the diseased agent

(pathogen) is one means of producing a recombinant vaccine.

10. Identifying a section of DNA that contains the required gene from the
source chromosome is the last stage in genetic engineering.

Guided Practice 2: Individual Work. You can choose only one item below
depending on your interest:

Item 1: Poster Making

Directions: The poster must illustrate, interpret, and emphasize the concept
of recombinant DNA technology. Upload your poster on your Facebook
account, tag your teacher with the hashtag
#GenBio2RecombinantDNATechnology.

Item 2: Educational Video Making

Directions: Create an educational video discussing the applications and
importance of recombinant DNA technology in a specific field (example:
medicine).
Guidelines:
A. Create your script and send it to your teacher for checking and
correction of information.
B. Create your 1-minute educational video and send it again to
your teacher for quality assurance.

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 C. Upload your quality assured video to your TikTok and
Facebook account. Tag your teacher with the hashtag
#GenBio2RecombinantDNATechnology.

(Note: Uploading your output on social media is OPTIONAL and will not affect
your score).

Your output will be evaluated using the 4-point rubric below:

RUBRICS for Guided Practice 1 (Poster Making and Educational Video

Making)
Scores
Categories 4 3 2 1

Relevance of All graphics All Most Most graphics do

Graphics to are graphics graphics not
the topic related to the are relate relate to the topic
topic related to to the
and make it the topic topic
easier to and most
understand make it
easier to
understand
Understanding Thorough Substantial Partial or Misunderstanding
of the and complete incomplete or misconception
Content
Accuracy Completely Generally Inaccurate Major
Accurate Accurate Inaccuracies
Attractive- Exceptionally Attractive Acceptably Distractingly
ness attractive in terms of attractive messy or
in terms of design, though it very poorly
design, layout, and may be a designed.
layout, and neatness. bit It is not
neatness. messy. attractive.
Originality Depicts an Not Not Not
important original, original. original. The
and original but it is Method of method of
message in a displayed the display is not
novel, in a novel display is creative
creative and creative, or original.
manner creative but
manner not novel.

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Total Points 25

Independent Practice: Reaction Paper

Directions: Read the news article below and write your comprehensive
reaction about it. You can use other sources of information related to the
topic.
Your reaction paper must include:
A. Introduction
B. Body
C. Conclusion
D. Citations and References (use APA 7th Edition)

Philippines Approves GMO Rice to Fight Malnutrition

By Steve Baragona
December 19, 2019

A breed of rice genetically engineered to combat vitamin A deficiency has

received approval from regulators in the Philippines.

Supporters say "Golden Rice" could remedy a condition that kills up to

250,000 children each year worldwide and blinds twice that number,
according to the World Health Organization.

It's the first genetically modified organism (GMO) designed to fight a public
health issue to get a green light from food safety officials in the developing
world.

Golden Rice has faced vigorous opposition from GMO opponents

throughout its development, citing safety concerns and other issues.
Protesters destroyed test fields in the Philippines in 2013.

The Philippine Department of Agriculture Bureau of Plant Industry

announced Wednesday that Golden Rice is as safe as conventional rice.
Regulators in the United States, Canada, Australia and New Zealand have
also cleared the grain of safety issues.

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 After 20 years of development, "it feels absolutely tremendous" to reach this
stage, said Adrian Dubock, Executive Secretary of the Golden Rice
Humanitarian Board, the nonprofit working to take the crop from the lab to
the field.

Two added genes turn rice golden, one from maize and one from a soil
bacterium. Under their direction, rice grains produce beta carotene, the
vitamin A precursor that makes carrots and sweet potatoes orange. A third
bacterial gene serves as a traceable marker.

In the Philippines, vitamin A deficiency among children has increased from

15.2% in 2008 to 20.4% in 2013, despite a national supplement program,
according to the Philippines-based International Rice Research Institute,
which is developing the crop.

Golden Rice could provide up to half of a young child's daily needs, IRRI
says.

Controversial crop

Biotech boosters have presented Golden Rice as one of the best examples
of what biotechnology can do, producing plants and animals that benefit
humanity faster than conventional breeding can.

Opponents have said the crops raise unknown risks, though the scientific
consensus is that GMO varieties on the market today are safe, including
Golden Rice.

GMO critics are also wary that the for-profit corporations that have
developed GMOs will have undue influence over the seed supply.

Agricultural biotech company Syngenta previously owned key patents for

Golden Rice but has donated them to the Golden Rice Humanitarian Board.
Dubock said Golden Rice strains are for use only by public and nonprofit
crop breeding programs and would not cost farmers any more than
conventional rice.

Dietary solution

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Critics say the considerable time, effort, and money spent on developing
Golden Rice would have been better spent pursuing efforts to diversify the
diets of the people who suffer from malnutrition.

"There are very limited funds available for development in third-world

countries. It really matters which route you choose to go, where you choose
to put your funds," said Bill Freese, a science policy analyst at the Center
for Food Safety.

Programs that get more fruits and vegetables into the diets of low-income
people would help alleviate several chronic ailments, not just vitamin A
deficiency, he noted.

Dubock agrees that "a diversified diet is the best solution," he said. But he
added that Golden Rice is a tool that works with how people are already
eating.

It's not clear when Philippine farmers will be able to grow Golden Rice.
Regulators still have to certify that the crop won't cause problems in
farmers' fields. IRRI says it will submit its application early next year.

Source: VOA News

Available at https://www.voanews.com/science-health/philippines-approves-gmo-rice-
fight-malnutrition

Your output will be evaluated using the 4-point rubric below:

RUBRICS for Guided Practice 2 (Reaction Paper)

Scores
Categories 4 3 2 1

Understanding Thorough Substantial Partial or Misunderstanding

of the topic and complete incomplete or misconception
Effective-ness Highly Generally Somewhat Ineffective
of reaction Effective Effective Effective
paper
Accuracy of Completely Generally Inaccurate Major
information Accurate Accurate Inaccuracies

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 Citation All dataMost data Some data Do not cite
obtained obtained obtained sources.
from otherfrom other from other
sources are sources are sources are
cited. Both cited. Both cited. Both
the text and the text and the text and
the the the
bibliography bibliography bibliography
used the APA used the used the
APA citation
citation style. APA citation
style style
Clarity of Exceptionally Generally Lacks Unclear
message clear clear clarity

Total Points 25

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 Assessment (1 point each)

Directions: Choose the letter of the correct answer. Write down your
responses on a separate sheet of paper.

1. It is a microorganism that lives in soil and makes proteins that are toxic to
some insects when eaten.
A. Escherichia coli C. Bacillus thuringiensis
B. Lactobacillus D. None of these

2. All of the following are structures of Plasmid DNA except:

A. Gene of interest C. Cilia
B. Origin of replication D. Antibiotic resistance gene

3. He is the first researcher to create a recombinant DNA molecule and shared

the Nobel Prize in Chemistry in 1980 for his contributions to recombinant
DNA technology.
A. Paul Berg C. James Watson
B. Gregor Mendel D. Francis Crick

4. Which bacterium is used to produce recombinant human insulin?

A. Bacillus thuringiensis C. Pseudomonas aeruginosa
B. Escherichia hermannii D. Escherichia coli

5. During the experiment, which site is used for the insertion of a gene of
interest?
A. Origin of replication C. Gene of interest
B. Restriction site D. Promoter site

6. In rDNA technology, which of the following enzymes is being used by

scientists to cut DNA molecules only in a particular area?
A. Isomerase C. Ligase
B. Restriction enzyme D. None of these

7. __________ is a small, double-stranded, circular DNA molecule that is

distinct from the chromosomal DNA of a cell.
A. Nucleoid C. Plasmid
B. Pili D. Plasma membrane

8. Modified Human Growth Hormone was made to address growth defects,

especially dwarfism. Which of the following gland is malfunctioning if a person
develops dwarfism?
A. Thyroid gland C. Pineal gland
B. Adrenal gland D. Pituitary gland

9. The following are examples of human antiviral recombinant vaccines

except:
A. Influenza A vaccine C. Polio vaccine
B. Insulin D. Hepatitis B vaccine

10. Which of the following is the correct sequence in genetic engineering?

1. Grow transformed cells to produce a GMO.
2. Identify a section of DNA that contains the required gene from the
source chromosome.
3. Insert plasmid into the host cell.
4. Extract the required gene.
5. Insert required gene into vector / bacterial plasmid.
A. 2, 4, 3, 5, 1 C. 4, 2, 3, 5, 1
B. 2, 4, 5, 3, 1 D. 2, 5, 4, 3, 1

Reflection

Directions: Answers must be precise and concise. You can use other sources
of information related to the topic. Write down your responses on a separate
answer sheet.
1. What is the role of recombinant DNA technology in improving life?
Elaborate on your answer.
2. How will knowledge of recombinant DNA technology be useful to resolve
the current challenges and issues we are facing today? Cite an example.

References

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“Bacillus Thuringiensis (Bt).” National Pesticide Information Center. Accessed
February 23, 2021.http://npic.orst.edu/

Baragona, Steve. “Philippines Approves GMO Rice to Fight Malnutrition”.

Science and Health. December 19, 2019.
https://www.voanews.com/science-health/philippines-approves-
gmo-rice-fight-malnutrition (accessed January 23, 2021).

Bawa, A. S., and K. R. Anilakumar. “Genetically Modified Foods: Safety, Risks

and Public Concerns—a Review.” Journal of Food Science and
Technology 50, no. 6 (2012): 1035–46.
https://doi.org/10.1007/s13197-012-0899-1.

Eldra P. Solomon, Linda R. Berg, and Diana W. Martin, General Biology,

Philippine Edition., (Philippines: Cengage Learning Asia Pte,Ltd., 2011),
215; 217-18; 233-34.

“Genetic Engineering.” Genome.gov. Accessed February 23, 2021.

https://www.genome.gov/genetics-glossary/Genetic-Engineering.

“Genetic Engineering.” Genome.gov. Accessed February 23, 2021.

https://www.genome.gov/genetics-glossary/Genetic-Engineering.

Nature News. Nature Publishing Group. Accessed February 23, 2021.

https://www.nature.com/scitable/definition/plasmid-plasmids-28/.

“Plasmid DNA- Structure, Function, Isolation And Applications.” Genetic

Education, January 10, 2020. https://geneticeducation.co.in/plasmid-
dna-structure-function-isolation-and-applications/.

“The Embryo Project Encyclopedia.” Golden Rice | The Embryo Project

Encyclopedia. Accessed January 23, 2021.
https://embryo.asu.edu/pages/golden-rice.
 Key to Corrections

Guided Practice 1: Assessment:

1. False (Correct Statement: Human insulin 1. C
provided by E. Coli was one of the first genetically 2. C
engineered proteins to be approved for human 3. A
use). 4. D
2. True. 5. B
3. True. 6. B
4. False (Correct Statement: The CGN-89564-2 7. C
genetically modified tomato was the first 8. D
genetically engineered crop product to be given a 9. B
human consumption license). 10. D
5. False. (Correct Statement: During the
experiment, the restriction site is used for the
insertion of a gene of interest.
6. False. (Correct Statement: Once metabolized
by the human body, the beta-carotene becomes
Vitamin A.
7. True.
8. True.
9. True.
10. False (Correct Statement:
A. Identifying section of DNA that contains the
required gene from the source chromosome is the
first stage in genetic engineering; or
B. Growing transformed cells to produce a GMO
is the last stage in genetic engineering.

Guided Practice 2: Answers may vary.

Independent Practice: Answers may vary.

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 Acknowledgment

The Schools Division of Zambales would like to express its heartfelt

gratitude to the following, who in one way or the other, have contributed to
the successful preparation, development, quality assurance, printing, and
distribution of the Quarter 2 Guided Learning Activity Kits (GLAKs) in all
learning areas across grade levels as a response to providing the learners with
developmentally-appropriate, contextualized and simplified learning
resources with most essential learning competencies (MELCs)-based activities
anchored on the principles of guided learning and explicit instruction:
First, the Learning Resources (LR) Development Team composed of the
writers and graphic artists for devoting much of their time and exhausting
their best efforts to produce these indispensable learning kits used for the
implementation of learning delivery modalities.
Second, the content editors, language reviewers, and layout evaluators
making up the Division Quality Assurance Team (DQAT) for having carefully
evaluated all GLAKs to ensure quality and compliance to DepEd standards;
Third, the Provincial Government of Zambales, for unceasingly
extending its financial assistance to augment the funds for the printing of
these learning resources for use by learners and parents at home;
Fourth, the teacher-advisers and subject teachers, in close
coordination with the school heads, for their weekly distribution and retrieval
of the GLAKs and for their frequent monitoring of the learners’ progress
through various means; and
Finally, the parents and other home learning facilitators for giving the
learners the needed guidance and support for them to possibly accomplish
the tasks and for gradually helping them become independent learners.
To deliver learning continuity in this challenging circumstance would
not be possible without your collective effort and strong commitment to
serving our Zambaleño learners.

Again, our sincerest thanks!

The Management Team

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