The document discusses genetic engineering, which involves manipulating DNA to transfer genes from one species to another, producing genetically modified organisms. Key processes include isolating the desired gene, selecting and inserting it into a vector, introducing the recombinant DNA into host cells, and mass culturing cloned genes for products like insulin. Various techniques and tools such as restriction endonucleases, vectors, and marker genes are utilized throughout this process.

1. Genetic Engineering
Anup Muni Bajracharya

2.  DNA - is the blueprint for the individuality of an organism.
 The life, growth and unique features of the organism depend on
its DNA.
 Genes - The segments of DNA which have been associated with
specific features or functions of an organism.
 Restriction endonucleases/ligases - Molecular biologists have
discovered many enzymes that can cut and join strands of DNA.
 Using such enzymes, scientists learned to cut specific genes from
DNA and to build customized DNA using these genes.
 Vectors - strands of DNA such as viruses, which can infect a cell
and insert themselves into its DNA.
 It can act as a carrier molecule for the transfer of genes into the
host.
Terms in Genetic engineering

3. What’s Genetic engineering???
a = bacterial cell.
b = cell’s main chromosome
c= plasmid
d= cut form of plasmid
e= restriction endonucleases
f= gene of interest like human
insulin gene
g=recombinant DNA
h= through a technique called
transformation, the genetically
engineered plasmid is re-inserted
into a bacterial cell.
i= mass cloning
j= Plasmid’s DNA will also be
transcribed and translated,
producing human insulin.
It involves direct manipulation of DNA, usually by introducing genes from one species
into another.

4. Some examples of Genetic
engineering- Glowing mice

5. Golden rice

6. Featherless chicken+ colourful cauliflower

7. Reduced fat in pork

8.  The process occurs in 5 steps as
 Isolation of desired gene or gene of interest
 Selection of vector and insertion of a gene of
interest into vector
 Introducing the r-DNA vector into host cells.
 Selection of appropriate host cell
 Mass culture of cloned genes
Steps in genetic engineering

9. Isolation of desired gene or gene of
interest

10.  Here the DNA or gene from an organism is isolated.
 That is from the whole genome obtained, the part of DNA
coding for the desired protein is isolated.
 This is a critical task and can be done by methods like
 i) Mechanical shearing. ii) Chemical synthesis.
 iii) By use of restriction endonucleases.
 iv) Complimentary DNA method.
 v) Gene bank
 The isolated genes are purified and taken for next step to
fix to a vector.
Isolation of the desired gene

11.  Mechanical shearing is
an experimental process
used to prepare DNA for
analysis or other
processing by the use of
mechanical instruments
to randomly cleave DNA.
 DNA is sheared to the
desired fragment range.
For instance, physical
shearing can be done by
probe sonication and
nebulization.
Mechanical shearing
probe sonication

12. Chemical synthesis

13. By use of restriction endonucleases

14. Complementary DNA

15. Gene bank

16.  Vector is a vehicle to carry the desired gene into the
genome of another organism.
Selection of vector

17.  These vectors have some specific properties like
 should be capable of independent multiplication.
This is possible if the gene has “Ori gene”
 should have a restriction site i.e. a site where the
isolated gene can be fixed using restriction
endonuclease. This is also called multiple cloning
sites.
 should have a gene promoter sequence like a β-
galactosidase gene.
 should have marker gene which helps to identify
transgenic cells.
Vector

18.  There are many types of vectors like
 a) Plasmids b) Cosmids
 c) Phasmid d) Transposons
 e) Bacteriophage (virus) f) Yeast
 g) Shuttle vectors
Types of vectors

19.  The isolated gene is now transferred into the vector.
 Restriction endonuclease enzyme is used to cut the
desired gene or gene of interest and same enzyme is
also used to cut the vector or plasmid.
 The cohesive or blunt ends are formed in vector to
which the gene of interest is attached or ligated using
DNA ligase enzyme.
Transfer of r-DNA:

21.  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 different methods like
 By use of CaCl2
 By electroporation
 By Transduction
Introducing the r-DNA into the host
cell.

22. By use of CaCl2

23. By Electroporation

26.  Once the transformation is done, now we need to
identify and isolate those bacteria from culture media
which have the vector within.
 So selection of those host cell consisting of our
desired gene should be selected.
 For this few methods are followed like
 Using of marker of gene technique
Selection of appropriate host cell:

27.  The marker gene is those which helps one to
know the bacteria which consists of desired
gene. For example, antibiotic resistance gene
(Tetracycline and Ampilcillin gene).
 The marker gene is present in the vector, one
of the marker gene let’s say tetracycline is cut
off by restriction endonuclease and gene of
interest is inserted there, the next one
ampicillin is free.
 Here the bacteria with the desired gene are
isolated on to media containing ampicillin.
 The bacteria with vector or rDNA has ampicillin
resistance gene so they multiply in the media
and form colonies. While all those without
vector do not grow and are inhibited.
Using of marker of gene technique

29.  The bacteria carrying the gene of interest is grown
massively with an aim to have the desired product in
huge quantity.
 For example, if insulin producing gene is incorporated
in bacteria then bacterial culture is done massively to
have desired product i.e insulin.
Mass culture of cloned genes

30. Thank you