Genetic engineering involves several key steps: 1) Isolating the desired gene, typically using restriction enzymes, 2) Inserting the gene into a vector like a plasmid for transfer, 3) Transforming the vector into a host organism like E. coli bacteria through processes like calcium chloride treatment, and 4) Identifying and isolating the transformed bacteria, usually using antibiotic resistance or phenotypic expression of the new gene. Common applications include producing insulin, growing human proteins in bacteria, and potentially modifying crops or curing genetic diseases.

1. RECOMBINANT DNA AND
GENETIC ENGINEERING

2. Melcs: Outline the processes involved in genetic
Engineering
Discuss the applications of recombinant DNA

3. AGREE/DISAGREE
Write AGREE if the statement is correct and DISAGREE if it
is wrong. Write your answer in the space provided for.
_______1. Cloning humans is legally accepted practice in the
Philippines.
_______2. E. Coli is one of the bacteria usually used in genetic
engineering.
_______3. Insulin is not a product of genetic engineering.
_______4. The first cloned pig is called Dewey.
_______5. Pharmaceutical world belongs to the red
biotechnology.
DISAGREE
AGREE
AGREE
DISAGREE
AGREE

4. GENETIC
ENGINEERING
Genetic engineering is
one of the most
controversial advances
in this age because of
some bioethical issues
that stick with it. Gene
splicing, cloning, and
test tube babies are
just some of the
products of research in
genetic engineering.

5. GENETIC ENGINEERING
 Genetic Engineering is the alteration of an
organism’s genotype using recombinant DNA
technology to modify an organism’s DNA to
achieve desirable traits. The addition of foreign
DNA in the form of recombinant DNA vectors
generated by molecular cloning is the most
common method of genetic engineering.
 The organism that receives the recombinant
DNA is called a genetically modified
organism (GMO)..

6. Stages:

7. EXAMPLE:

8. In most cases, the desired organism is human or other
organisms of human interest. While the organism of
choice is mostly bacteria or yeast.
But why only bacteria and yeast? Because they can be
quickly grown and also their life cycle completes in a
few hours to days. Due to this, we get the desired
product formed in a short time. Because of such a
short lifespan, they express the transferred gene to the
fullest and we obtain the product very fast.


9. STEPS OF GENETIC ENGINEERING
1. Isolation of the desired gene
Here the DNA coding for the desired protein is
isolated. This is a critical task and can be done
by any of the following four methods like;
1.Mechanical shearing.
2.Chemical synthesis.
3.By the use of restriction endonucleases.
4.Complimentary DNA method.

10. ISOLATION OF THE DESIRED GENE
Mechanical shearing
 Here the required gene is cut
off from the whole gene by
use of mechanical force. This
can be done by methods like
sonication, nebulization,
point Shink shearing, needle
shear, etc. This method leads
to the formation of random
DNA fragments.
Chemical synthesis
 As the name indicates, here
the desired gene is
synthesized by the use of
free nucleotides. For this, the
target protein is isolated and
from it, the required
nucleotide sequence is
deduced.

11. ISOLATION OF THE DESIRED GENE
Using restriction
endonuclease enzymes
 Using restriction endonuclease
enzymes
 In this method, the whole
genome is taken and subjected
to the enzyme restriction
endonucleases. This enzyme
cuts the DNA at specific points
like the scissors. The gene
obtained by this is quite perfect
and hence widely used.
Complimentary DNA
method
 Here the desired DNA
sequence is synthesized
from the messenger RNA
which codes of the specific
protein of choice. For this,
the enzyme reverse
transcriptase is used to
synthesize the double-
stranded DNA sequence.

12. STEPS OF GENETIC ENGINEERING
2. Selection of vector
A vector is a vehicle to carry the desired gene into the
genome of another organism. This helps us to see that
the gene is not destroyed during transfer. Also, the gene
will be operational inside the new organism due to the
vector.

13. TYPES OF VECTORS
1.Plasmids: These are naturally occurring
proteins from bacteria.
2.Cosmids:
3.Phasmid
4.Transposons
5.Bacteriophage (virus)
6.Yeast
7.Shuttle vectors:

15. A bacteriophage is a virus that attacks bacteria and
inserts its gene into the bacterial cell for
multiplication. Cosmid is similar to plasmid DNA but
can accommodate large DNA pieces.
Transposons: These are movable genes or jumping
genes which move from one cell to another or
plasmid to the nucleus. The size is very small like
1kb to 2kb (1kb =1000nucleotide). This transposon
has no “marker gene” and “ori gene.”
Yeast cloning vector: These are used to transfer the
desired gene into fungi. This is a similar plasmid
with little modification.
Shuttle vector: These vectors have ori-gene,
promoter gene for both bacteria and fungi. So, it is
two in one type of process.

16. STEPS OF GENETIC ENGINEERING
3. Transfer of rDNA
The isolated gene is now transferred into the vector in
this step.

17. TRANSFER OF RDNA
Cohesive technique
 Here cohesive ends are formed
for joining with the vector.
Restriction endonuclease
enzyme is used to cut the
desired gene and also plasmid.
By these cohesive ends are
formed. These cohesive ends in
both plasmid and the desired
genes are easily attachable.
Homopolymer chain
 Here polymers are
formed at the ends of the
gene to fix with the
vector.

18. TRANSFER OF RDNA
Blunt end joining
 Here the genes with blunt
ends are joined to vector by
use of DNA ligase enzyme.

Use of Cos sites.
 Cos site is one that has 12
nucleotide chains. The vector
with the gene is transferred
into a bacteriophage. As we
know, the bacteriophage is a
virus that attacks bacteria
and multiplies. So,
bacteriophages transfer the
desired gene loaded vectors.


19. STEPS OF GENETIC ENGINEERING
4. Transformation of rDNA
Here the vector with the tagged desired gene is
transferred into the organism of interest, i.e.,
bacteria or fungi in most cases. This is done by
creating holes in the bacterial cell wall. For this,
we use two methods

20. TRANSFORMATION OF
RDNA
By use of CaCl2
 Here bacteria and calcium chloride are
taken in a Petri dish and cooled to 0-4
degrees. Then rDNA is added and the
temperature is suddenly raised to
42degree. When cooled the bacterial
wall shrinks and when heated
instantly, it expands abnormally
creating pores in the wall. The loaded
vector enters the cell through these
pores.
By use of lysosomal
enzymes
 This lysosomal enzyme
destroys bacterial cell walls.
So, this catalytic enzyme is
taken in low concentration
along with plasmids (vector)
and added to the bacterial
culture. The cell wall cracks
and plasmids enter.

21. STEPS OF
GENETIC
ENGINEERING
5. Identification,
isolation & culture
of transgenic
bacteria
 Once the transformation
is done, now we need to
identify and isolate
those bacteria from
culture media that have
the vector within.

22. IDENTIFICATION, ISOLATION &
CULTURE OF TRANSGENIC
BACTERIA
Antibiotic sensitivity
technique
 This is based on the replica plating
method. Here the bacteria with the
desired gene are isolated on to another
media. For this, the solution of
bacteria is taken and added with
antibiotic ampicillin. Those with
ampicillin resistance genes multiply.
While all those without vector do not
grow and are inhibited. The remaining
ones grow into visible colonies.

Direct phenotypic
identification
 Here transgenic bacteria are
identified based on the newly
developed characters. For
example, bacteria with β-
lactamase producing gene
survive the culture media
when added with ampicillin
while remaining die.