Republic of the Philippines

Department of Education
REGION I
SCHOOLS DIVISION OF THE CITY OF BATAC

ACTIVITY SHEETS IN GENERAL BIOLOGY II

QUARTER 3, WEEK 1

GENETIC ENGINEERING AND

RECOBINANT DNA APPLICATIONS

MELCs: 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:
1. Compare classical breeding with modern genetic engineering techniques.
2. Describe some methods to introduce DNA into cells.
3. Explain the selection and screening of transformants / genetically modified
organisms (GMOs).
4. Give examples of products from recombinant DNA technology.
5. Illustrate the use of databases to search genes for desired traits.
6. Describe steps in PCR to amplify and detect a gene of interest.
7. Explain how genes may be cloned and expressed.

Prepared by:

PRINCE KEVIN P. ADINA

Special science Teacher I
GENETIC ENGINEERING

In order to survive, man has successfully domesticated selected plants and

animals. He has taken an active part in choosing desired traits of plants and animals.
Traits that were considered valuable (i.e. high fruit yield; high milk production, etc.)
were sought out and propagated. The processes involved may include classical
breeding practices such as controlled pollination of plants, and the mating of animals
with desired traits. In today’s modern science, molecular biology techniques are being
employed in the insertion and expression of proteins in different organisms for various
purposes.

ENHANCE TRAIT MODIFYING TECHNIQUE

Kobe / Wagyu Beef (Beef with good fat
Classical breeding
distribution)

Guapple (Large sized guava) Classical breeding

Human Insulin-producing bacteria Recombinant DNA Technology

Flavr-Savr (Delayed-ripening tomatoes) Recombinant DNA Technology

Macapuno trait in coconuts Classical breeding

• Classical breeding practices focus on the mating of organisms with desirable

qualities.

• Genetic engineering involves the use of molecular techniques to modify the traits
of a target organism. The modification of traits may involve:
a. introduction of new traits into an organism
b. enhancement of a present trait by increasing the expression of the desired
gene
c. enhancement of a present trait by disrupting the inhibition of the desired
genes’ expression.

• General outline of recombinant DNA can be observed 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 ( e.g. circular plasmid in bacteria with a foreign gene of
interest)
3. ligation (join together) of the gene of interest (e.g. 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

• Different ways of introducing plasmids to host organisms.

Biolistic. 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 pretreatment (usually with CaCl 2) 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
“Heat Shock” 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.

• Some methods to screen recombinant cells are as follows:

Selection of plasmid DNA containing cells
A selection marker within the inserted plasmid DNA sequence allows the
selection of “transformants”. Usually, an antibiotic resistance gene (e.g. AMP
ampicillin resistance gene) is included in the plasmid DNA. This allows only
“transformed” cells to survive in the presence of the antibiotic (e.g. ampicillin).
Plating the plasmid-cell solution on antibiotic containing media will select for these
“transformants” and only allow plasmid-containing cells to grow and propagate into
colonies.

Selection of transformed cells with the desired gene

Certain inserted genes within the plasmids provide visible proof of their
presence. These include the antibiotic resistance genes that allow for the selection
 of the transformed cells within the solution. Some inserted genes also produce
colored (e.g. chromogenic proteins) or fluorescent products (e.g. GFP) that label
the colonies/cells with the inserted gene.
In some cases, the location of the cloning site within the plasmid is in the
middle of a gene (i.e. β-galactosidase, lacZ) that generates a (blue) colored product
in the presence of a substrate (i.e. isopropyl β-D-1 thiogalactopyranoside, or IPTG).
Cells transformed with these “empty” plasmids will turn blue in the presence of
IPTG. Insertion of a gene in the cloning site disrupts the sequence of the β-
galactosidase gene and prevents the generation of the colored product in the
presence of the substrate. Cells transformed with the disrupted β-galactosidase
gene will remain “white” in the presence of IPTG. This “blue-white screening”
protocol is thus able to screen for cells that were transformed with the desired gene
in the cloning site.
PCR detection of plasmid DNA
Alternatively, the presence of the desired gene in the inserted plasmids may
be confirmed using PCR amplification. PCR reactions specific for the desired gene
may be done using DNA from cells. Amplification of the expected product would
confirm the presence of the gene within the samples. PCR reactions specific for
plasmid sequences will also confirm/identify the type of plasmid used for the
transformation.
Agarose gel electrophoresis (AGE) allows the identification of PCR
products and estimation of their sizes. This is done by running a molecular weight
(MW) ladder alongside the samples. The MW ladder is made up of DNA fragments
of known size (e.g. 100 base pairs, 200bp, 300bp, 500bp, etc). The size of the PCR
product may be approximated by the DNA fragment in the MW ladder that runs a
similar distance.

• Genetically Modified Organisms (GMOs)

With the ability to insert gene sequences, comes the possibility of providing
new traits for these target organisms. This has allowed the development of GMOs.
Some of these genetic modifications promise higher product yield for their targets.
These include the Flavr-Savr Tomato and Bt-Corn.

The Flavr-Savr (“Flavor Savor”) tomato was the first genetically modified
organism that was licensed for human consumption. The trait modified in this
tomato is its ripening process. A gene for an enzyme that causes the degradation
of pectin in the cell walls (i.e. polygalacturonase) normally softens the fruit as it
ripens. In Flavr Savr tomatoes, an inhibitor (i.e. antisense RNA) disrupts the
expression of this gene, thereby delaying the softening of the fruit and extending
the time it may be kept in storage and transported to markets.
Bt-Corn was developed to incorporate the production of a toxin (i.e. Bt-
endotoxin) from Bacillus thuringensis in corn plants. This toxin results in the death
of pests that feed on these plants like the corn borer larvae. The toxin has been
 shown to be selective for Lepidoptera larvae and is non-toxic to humans,
mammals, fish and birds. The selective toxicity of the toxin allows its use in food
crops. The introduction of the toxin is believed to increase crop production due to
decreased losses from pest infestation. The same technology has been applied in
the Philippines for the development of Bt-Eggplant.
Despite the proposed benefits of GMOs, some people have raised their
concerns regarding the consumption of these modified foods. While most of the
products are tested for safety, concerns are raised for the possibility of not being
able to detect hazards that are present, but are currently undetectable by today’s
current technology.
Because of these issues, manufacturers are urged to provide labels that
notify consumers of GMO presence in their products. While GMOs are believed to
be safe when licensed by the food regulatory agencies, it is believed that the
consumers must be provide with enough information to make their own choices
regarding their use.

APPLICATIONS OF RECOMBINANT DNA

The Central Dogma of Molecular Biology

DNA (gene) → RNA (transcript) → Protein (trait)

Different organisms have different traits based on their genes (DNA

sequences). For example, frogs have antimicrobial peptides on their skin. Some
jellyfish have proteins that allow them to glow in the dark. Mutations in hemoglobin
genes lead to anemia.
Based on the central dogma, if transcription and translation of genes lead to
some traits, then the insertion of certain genes in a given organism may provide it with
new traits. This is the basis for the development of genetically modified organisms
(GMOs).
Bear in mind that for a gene to add a trait to an organism, the gene for the trait
must be inserted within the target organism, and the organism should ha v e the
necessary “equipment” (i.e. enzymes, materials ) to produce the protein that results in
the trait or desired phenotype.

PCR Amplification
Once a desired trait is chosen, information must be acquired for either its
detection or expression in a given organism.

1. Detection
Some researchers may be interested in determining if a given gene/trait
is available in a particular organism. If no previous research provides this
information, researchers may test the DNA of different organisms for the
presence of these specific genes. A technique that allows the detection of
specific genes in target organisms is called PCR.
PCR amplification is an in-vitro method that simulates DNA replication
in vivo. It utilizes a thermostable (heat-resistant) DNA polymerase that builds
single stranded DNA strands unto unwound DNA templates. PCR uses
repeated cycles of incubation at different temperatures to promote the
unwinding of the DNA template (~95°C); the annealing of a primer (a ~20bp
oligonucleotide sequence (recall RNA primers in DNA replication) onto the
ssDNA template strand (~54 - 60°C); and the extension of the generated
ssDNA strand through the binding of complementary bases to the template
strand (~72° C). The thermostability of the polymerase allows it to survive the
repeated cycles of denaturation, annealing and extension with little loss of
enzyme function. Each cycle of PCR doubles the amount of the target
sequence. A typical PCR experiment uses about 35 cycles of amplification. This
increases the original amount of the target sequence by 235 (i.e. ~34 billion)
times.
Gene detection by PCR involves the design of primers that would only
bind to sequences that are specific to a target. For example, researchers would
want to find out if gene X (e.g. the gene for insulin) is available in a target
organism (e.g. a mouse, Mus musculus). Primers may be designed by looking
at the available sequences for gene X in the databases (e.g. all the genes for
insulin in different organisms; humans, pigs, cows, etc.). The different gene X
sequences must be aligned/compared to match areas of sequence similarity
(conserved sequences) and areas of sequence dissimilarity (non-conserved
sequences). Primers designed to have the same sequence as the conserved
areas will be specific for binding gene X sequences in all the target organisms.
Primers designed to have the same sequence as the non-conserved areas will
only be specific for the organisms which match its sequence.
Unlike DNA replication in vivo, PCR reactions do not use too many
helper enzymes such as helicases and gyrases to help denature and stabilize
the template DNA strands. The cyclic heating of the samples is meant to
provide the physical separation of the template DNA strands through heat
denaturation of the inter-strand H-bonds.
To better understand the different steps in PCR amplification, you can
watch this video on how a specific target DNA can be amplified through PCR.
https://www.youtube.com/watch?v=iQsu3Kz9NYo

PCR Applications
PCR may be used to detect the presence of a desired gene in an
organism. Depending on the primer design, the expected product may
represent only a specific region of the gene or the entire gene itself. The first
case is useful for detection of the gene, or the detection of organisms with that
specific gene within a sample. The second case is useful for the amplification
 of the entire gene for eventual expression in other organisms. The direct
amplification/copying of a full gene is part of the process for “cloning” that gene.

2. Cloning and Expression

Some genes provide economically, and industrially important products
(e.g. insulin-coding genes; genes for collagen degradation). In some cases,
scientists would want to put these genes into organisms for the expression of
their products. One example would be the insertion of an insulin coding gene
from the human genome into bacteria. This allows the “transformed” bacteria
to now produce human insulin as a product.
Certain types of bacteria are capable of this process since they are able
to take genes within their cell membranes for eventual expression. The genes
are normally in the form of small, circular DNA structures called plasmids. The
genes found in the inserted plasmid DNA sequence will be expressed as
proteins that provide specific traits to the transformed bacteria.

The following table shows examples of modified traits using cloned genes and their
applications:

Recipient
Modified Trait Gene Modification Application (Field)
Organism
(Medicine) Production of
Insertion of Human
Insulin Production Bacteria Human Insulin in
Insulin Gene
Bacteria
(Agriculture)
Insertion of Bt-toxin Production of corn plants
Pest Resistance Corn / Maize
gene with increased
resistance to corn boxer
Agriculture)
Production of plants with
fruits that have delayed
Disruption of a
ripening fruits. These
gene for a ripening Tomato
Delayed Ripening fruits will survive longer
enzyme (e.g. Plant
transport time, allowing
polygalacturonase)
their delivery to further
locations (i.e. export
deliveries)
(Industry)
Enhance large scale
Chymosin Insertion of a gene
Bacteria production of chymosin.
Production for chymosin
This enzyme serves as a
substitute for rennet in
the coagulation of milk.
Rennet has to be
harvested from calves.
The large scale
production of this
enzyme in bacteria
provides an abundant
supply of this important
component for the
cheese
production industry.
Title of the Activity: Modifying Traits – Recombinant DNA Technology vs.
Classical Breeding

Direction: Complete the Venn Diagram below differentiating and

comparing classical breeding and Recombinant DNA Technology
as modifying technique in enhancing traits of an organism.

CLASSICAL BREEDING RECOMBINANT DNA

TECHNOLOGY
Name: ___________________________________________Date: _____________

Grade/Section: ____________________________________ Score: ____________

Title of the Activity: Designer Genes

Directions: Read the following scenario and come up with a

“designer gene” that could possibly be beneficial. Use the succeeding
page as your answer sheet.

Imagine that you are a geneticist and you are tasked at constructing a genetically
modified organism/trait. In doing so, you need to identify some things such as the
following:
a. Identify a special trait (e.g. large fruit size)
b. Identify a source organism of that trait (e.g. jackfruit/langka)
c. Identify a target organism where you can apply the trait (e.g. aratilis)
d. Identify the product organism; modified / added trait (e.g. langka-sized aratilis)

Give a brief discussion of the “designer gene” you’ve come up with. Consider the
following criteria in coming up with your designer gene.
1. Originality of the study (Has anyone done studies of this type before?)
2. Feasibility of the study (How possible is the proposed modification? Can the
target organism support the proposed trait?)
3. Potential applications of the new organism (What benefits would the
recombinant organism provide to society?)

You can have the following as an example: Flood-resistant rice; Delayed ripening of
fruits.
Trait:

Source Organism:

Target Organism:

Product:

Description:
Title of the Activity: Solidifying Your Ideas – Procedures and Methods in
Recombinant DNA Technology

Directions: Make a digital poster on the steps and other methods

involved in recombinant DNA. Use an A4 size bond paper for your
output. Criteria in creating your digital poster is provided below as
your reference.

You will be assessed based on the following criteria:

Criteria 10 points
Content The output exceeds the expectations.
Correctness The output is free from errors.
Creativity and The work demonstrates superior creativity and
Originality originality in the selection of the visuals.

Title of the Activity: Applying Concepts in Processes of Genetic Engineering

and Recombinant DNA

Directions: Provide a substantial answer to the following. Write

your answer in the answer sheet provided. Refer to the criteria
given as your guide in constructing your output.

How do you think does PCR may be used for the detection of disease causing
pathogens in a population?

For example, it may be used to check if a patient has dengue virus infection. This is
done by using primers that are specific for complementary DNA (cDNA) sequences
that correspond to the dengue virus. If PCR amplification occurs using cDNA from a
patient’s blood samples then the patient likely has dengue viruses in his/her blood.

With our current situation, discuss how PCR can be used for the detection of SARS-
COV-2 that causes COVID-19.

You will be assessed based on the following criteria:

Criteria 10 points
Content The output exceeds the expectations.
Correctness The output is free from errors.
Discuss how PCR can be used for the detection of SARS-COV-2 that causes
COVID-19.
References

Department of Education Central Office. Most Essential Learning Competencies

(MELCs). 2020.

Teaching Guide for Senior High School GENERAL BIOLOGY 2. PDF. Quezon City:
Commission on Higher Education, 2016.
ANSWER KEY:

Title of the Activity: Designer Genes

➢ Answers may vary

Title of the Activity: Modifying Traits – Recombinant DNA

Technology vs. Classical Breeding

➢ Answers may vary

Title of the Activity: Solidifying Your Ideas – Procedures and

Methods in Recombinant DNA Technology

➢ Answers may vary

Title of the Activity: Applying Concepts in Processes of Genetic

Engineering and Recombinant DNA

➢ Answers may vary