Hello, learners!

Welcome to
your first lesson, which is about
Genetic Engineering. Here, you
will be
able to learn the historical
developments of genetic
engineering, its processes and
techniques, and
how it works; you will also
learn molecular cloning,
genetically modified organisms
and recombinant
DNA applications. Are you
excited? Come on, let's get
started
Hello, learners! Welcome to
your first lesson, which is about
Genetic Engineering. Here, you
will be
able to learn the historical
developments of genetic
engineering, its processes and
techniques, and
how it works; you will also
learn molecular cloning,
genetically modified organisms
and recombinant
DNA applications. Are you
excited? Come on, let's get
started!
Hello, learners! Welcome to
your first lesson, which is about
Genetic Engineering. Here, you
will be
able to learn the historical
developments of genetic
engineering, its processes and
techniques, and
how it works; you will also
learn molecular cloning,
genetically modified organisms
and recombinant
DNA applications. Are you
excited? Come on, let's get
started!
Hello, learners! Welcome to your first lesson, which is about Genetic Engineering. Here, you will be able
to learn the historical developments of genetic engineering, its processes and techniques, and how it
works; you will also learn molecular cloning, genetically modified organisms and recombinant DNA
applications. Are you excited? Come on, let's get started!

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.

Before we go further, let us check your knowledge of genetically modified organisms. Enumerate plants
and animals that have desirable or enhanced traits.

Traits that were considered valuable 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.

Over the last 50 years, the field of genetic engineering has developed rapidly due to a greater
understanding of deoxyribonucleic acid (DNA) as the chemical double helix code from which genes are
made. The term “genetic engineering” is used to describe the process by which the genetic makeup of
an organism can be altered using “recombinant DNA technology.” This involves the use of laboratory
tools to insert, alter, or cut out pieces of DNA that contain one or more genes of interest. It is sometimes
called “genetic modification.”

The modification of traits may involve:

 Introduction of new traits into an organism

 Enhancement of a present trait by increasing the expression of the designed gene

 Enhancement of a present trait by disrupting the inhibition of the desired genes’ expression

Historical Developments

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 them are
artificial insemination, in vitro fertilization (e.g., “test-tube” babies), cloning, and gene manipulation. In
the latter part of the 20th century, however, the term came to refer more specifically to methods of
recombinant DNA technology (or gene cloning), in which DNA molecules from two or more sources are
combined either within cells or in vitro and are then inserted into host organisms in which they are able
to propagate.

The possibility of recombinant DNA technology emerged with the discovery of restriction enzymes in
1968 by Swiss microbiologist Werner Arber. The following year, American microbiologist Hamilton O.
Smith purified the so-called type II restriction enzymes, which were found to be essential to genetic
engineering for their ability to cleave a specific site within the DNA (as opposed to type I restriction
enzymes, which cleave DNA at random sites).

Drawing on Smith’s work, American molecular biologist Daniel Nathans helped advance the technique
of DNA recombination in 1970–71 and demonstrated that type II enzymes could be useful in genetic
studies. Genetic engineering based on recombination was pioneered in 1973 by American biochemists
Stanley N. Cohen and Herbert W. Boyer, who were among the first to cut DNA into fragments, rejoin
different fragments, and insert the new genes into E. coli bacteria, which then reproduced.

Process and Techniques Most recombinant DNA technology involves the insertion of foreign genes into
the plasmids of common laboratory strains of bacteria. Plasmids are small rings of DNA; they are not
part of the bacterium’s chromosome (the main repository of the organism’s genetic information).
Nonetheless, they are capable of directing protein synthesis, and, like chromosomal DNA, they are
reproduced and passed on to the bacterium’s progeny. Thus, by incorporating foreign DNA (for example,
a mammalian gene) into a bacterium, researchers can obtain an almost limitless number of copies of the
inserted gene. Furthermore, if the inserted gene is operative (i.e., if it directs protein synthesis), the
modified bacterium will produce the protein specified by the foreign DNA.

Figure 1: Plasmids structure of bacteria

A subsequent generation of genetic engineering techniques that emerged in the early 21st century
centered on gene editing. Gene editing, based on a technology known as CRISPR-Cas9, allows
researchers to customize a living organism’s genetic sequence by making very specific changes to its
DNA. Gene editing has a wide array of applications, being used for the genetic modification of crop
plants and livestock as well as laboratory model organisms (e.g., mice). The correction of genetic errors
associated with disease in animals suggests that gene editing has potential applications in gene therapy
for humans.

How does genetic engineering work?

a. To help explain the process of genetic engineering, we have taken the example of insulin, a protein
that helps regulate the sugar levels in our blood.

 Normally, insulin is produced in the pancreas, but in people with type 1 diabetes, there is a problem
with insulin production.

 People with diabetes therefore have to inject insulin to control their blood sugar levels.

 Genetic engineering has been used to produce a type of insulin, very similar to our own, from yeast
and bacteria like E. coli.

 This genetically modified insulin, ‘Humulin’ was licensed for human use in 1982.

b. The genetic engineering process:

1. A small piece of circular DNA called a plasmid is extracted from the bacteria or yeast cell.
2. A small section is then cut out of the circular plasmid by restriction enzymes, ‘molecular scissors.’

3. The gene for human insulin is inserted into the gap in the plasmid. This plasmid is now genetically
modified.

4. The genetically modified plasmid is introduced into a new bacteria or yeast cell.

5. This cell then divides rapidly and starts making insulin.

6. To create large amounts of the cells, the genetically modified bacteria or yeast are grown in large
fermentation vessels that contain all the nutrients they need. The more the cells divide, the more insulin
is produced.

7. When fermentation is complete, the mixture is filtered to release the insulin.

8. The insulin is then purified and packaged into bottles and insulin pens for distribution to patients with
diabetes.

Figure 2: An illustration how genetic modification is used to produce insulin in bacteria.

A general outline of recombinant DNA:

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

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

3. (e.g. circular plasmid in bacteria with a foreign gene of interest)

4. Ligation (joining together) of the gene of interest (e.g. from an animal) with the vector (cut bacterial
plasmid)

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

6. Selection process to screen which cells actually contain the gene of interest
7. Sequencing of the gene to find out the primary structure of the protein

Methods to introduce DNA into cells:

 Biolistic – in this technique, a “gene gun” is used to fire DNA-coated pellets onto plant tissues. Cells
that survive the bombardment are able to take up the expression plasmid-coated pellets and acquire
the ability to express the designed protein.

Figure 3: Plant Transformation using Particle Bombardment

 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 CaCl2) is said to make the cells
“competent” to accept the plasmid DNA. After the cells are made competent, they are incubated with
the desired plasmid at about 4 degrees Celsius for about 30 minutes. 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 degrees Celsius for 1 minute, then back at 4 degrees Celsius for 2 minutes. The rapid rise and drop in
temperature is believed to increase and decrease the pore sizes in the membrane. The plasmid DNA
near the membrane surface is 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 the insertion of genes into mammalian cells.

Methods to screen recombinant cells:

 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.
Figure 4: Plasmid Map

 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 that
generates a (blue) colored product in the presence of a substrate. 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 beta-galactosidase gene and prevents the generation of the colored product in the
presence of the substrate. Cells transformed with the disrupted beta-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 at 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 maybe 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.

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 yields for their targets. These include the Flavr-Savr Tomato and Bt-Corn.

a. 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.

b. Bt-Corn was developed to incorporate the production of a toxin (i.e. Bt-endotoxin) from Bacillus
thuringiensis into 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.

Figure 5: Flavr-Savr tomato (left) and Bt corn (right)