Genetic Engineering – Gen

Biology 2
LEARNING OBJECTIVES:

1. Outline the processes involved in genetic engineering (STEM_BIO11/12-IIIa-b-

6)

2. Discuss the applications of recombinant DNA. (STEM_BIO11/12-IIIa-b-7)

The central Dogma of molecular biology explains the flow of genetic information
and the molecular mechanism in understanding how genotype translate to
phenotype, it became apparent then that changing an organismal trait is
possible by altering its genetic make-up known as genetic engineering.

You have learned already from your grade 8 Biological science about the
Central Dogma of Molecular Biology which covers the lesson on nucleic acid
and its types- DNA and RNA and their structures and the flow of genetic
information from genes to proteins through the process of DNA replication,
transcription, translation in order to make an enormous variations of proteins.
You have learned also about heredity and variation wherein traits are being
inherited by the off springs from their parents and that desirable and
undesirable traits of an off springs including diseases can be inherited from
their parents. But the possibility to enhance a good trait will be possible now
through the process of genetic engineering.

Many people are unaware that humans have been practicing genetic
engineering since the ancient times. Selective breeding or classical breeding in
agricultural crops and livestock has actually altered the genetic make-up 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.

Genetic engineering involves 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 as to enhancement of present

traits by increasing the expression of the desired gene or by disrupting the
inhibition of the desired genes’ expression.

Genetic engineering includes classical breeding which is considered as the

traditional way of genetic engineering which practices the mating of organisms
with desirable qualities and Recombinant DNA technology (rDNA), a modern
technique of genetic engineering.

The general outline of recombinant DNA are as follows;

I. Cutting or cleavage of DNA by restriction enzymes (REs) as shown on steps 2
on the diagram. Restriction enzymes are called ‘molecular scissors’ cutting the
DNA at specific target sequences leaving a single-stranded overhang at the site
of the cleavage (step 2). These overhangs of the donor DNA (gene of interest)
will be paired with other overhangs (vector DNA).

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

recombinant DNA (shown on step 1). The most commonly used as vectors are
plasmids (circular DNA molecules that originated from bacteria, viruses and
yeast cells). Plasmids are not part of the main cellular genome, but they carry
genes that provide the host cell with useful properties such as drug resistance,
mating ability, and toxins production. They are small enough to be conveniently
manipulated experimentally and furthermore, they will carry extra DNA that is
spliced to them.

III. Ligation (join together) of the gene of interest (eg. from animal) with the
vector (cut bacterial plasmid) as shown on step 3 of the above diagram.

Recombinant DNA technology is the joining together of DNA molecules from

two different species. The recombined DNA molecule is inserted into a host
organism to produce new genetic combinations that are of value to science,

medicine, agriculture, and industry

The resulting molecule is called recombinant DNA. It is recombinant in the

sense that it is composed of DNA from two different sources.
IV. Transfer of the recombinant plasmid into a host cell (that would carry out
replication to make huge copies of the recombined plasmid). In the above
diagram as shown in steps 4, the host cell is a bacterium known also as
recombinant bacterium which will undergo cloning or replication of recombinant
DNA

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

interest.

VI. Sequencing of the gene to find out the primary structure of the protein.
Once a segment of DNA has been cloned, its nucleotide sequence can be
determined. The nucleotide sequence is the most fundamental level of
knowledge of a gene or genome. It is the blueprint that contains the instructions
for building an organism, and no understanding of genetic function or evolution
could be complete without obtaining this information.

After the process of recombinant DNA, these plasmids or gene copies will now be
introduced to its host organisms to confer upon them the desired trait. A gene
for pest resistance for example, as shown from the image above, may be
isolated, cloned and inserted into plant cell. Alternatively, bacterial cells may
express the inserted gene in order to produce protein products. Some important
human proteins like hormones and enzymes are produced by this technique.

These are some ways in which these plasmids may be introduced into host
organisms;

1. 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.

2. 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” 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
The next step after cloning, therefore, is to find and isolate that clone among
other members of the library. If the library encompasses the whole genome of an
organism, then somewhere within that library will be the desi

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”.

3. 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.

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

Genetically modified organisms (GMOs) have been produced through the rDNA
technology technique. Genetically modified plants are produced by integrating a
gene of interest into the Ti-plasmid before inserting the plasmid into the plant
cells. These now possess gene that would confer traits such as resistance to
certain bacterial or fungal pests. For example, genetically engineered corn also
called as ‘Bt corn’ expresses a gene from the soil-dwelling bacterium Bacillus
thuringiensis, making
them resistant to the corn borer disease. The ‘golden rice’ is a transgenic variety
of rice that is engineered to produce beta-carotene and prevent Vitamin-A
deficiency. Rice and potato have been modified to produce harmless proteins
derive from cholera to serve as a natural vaccine. Soybean has been engineered
to have resistance against herbicides.

Genetic modification is not only for plants. Recombinant bacterial cells with
human genes can be used in order to produce human proteins like insulin to be
used by people with type I diabetes, human growth hormones which is taken to
cure stunted growth, and tissue plasminogen activator which dissolves blood
clots among patients who had heart attack. If larger quantities of these protein is
required, an option is to insert the gene in animals. For example, transgenic pigs
to produce human hemoglobin, transgenic goat for human clotting factor and
other transgenic animals that is used to synthesize pharmaceutical products
referred as ‘pharm animal’.