1

GOVT. ARTS & SCIENCE COLLEGE RATLAM

Topic : GENETIC ENGINEERING

Submitted to: Submitted by:Ms Kanushri Ranawat Shivangi Soni M.SC FINAL BIOTECHNOLOGY

2

INDEX

• Introduction• Steps involved in Genetic Engineering • Tools used in Genetic Engineering Gene of interest (DNA insert) Restriction Enzymes Vector Host cell• Gene transfer methods• In vitro production of Insulin• Transgenic plant• Transgenic animal• Application

3

HISTORY

Father of Genetic Engineering is Paul Berg. He was the first who developed recombinant DNA technology.

4

THE BEGINNING

• The first genetically modified animal was mouse created in 1973 by Rudolf Jaenisch.

• In 1993, an antibiotic resistant gene was inserted in tobacco plant, leading to first genetically modified plant.

• In 1978, the technology was commercialized with the production of insulin.

• In 1994, first genetically modified food Tomato was made.

5

DEFINATION

The change in genetic make up of living cells by inserting desired gene through a vector in called

genetic engineering (GE).

6

TERMS

• Gene: The gene is small piece of DNA that encodes for a specific protein.

• Recombinant DNA: The DNA formed by joining DNA segment of two different organism.

• Recombinant DNA technology: The technique by which gene of interest is transferred to the host.

• Genetically modified organism: The organism whose genetic make up is altered/changed using rDNA technology.

7

PROCEDURE

• Isolation of desired DNA fragment(gene of interest) with the help of restriction enzymes.

• Isolation of DNA vector.• Construction of rDNA. In this gene of interest is

inserted into the vector.• Introduction of vector containing recombinant into

the host cell.• Multiplication of Host cells containing recombinant

DNA.• Expression of cloned gene.• Selection of Recombinant cells.

8

9

Selection of transformed cell

10

TOOLS USED IN GENETIC ENGINEERING

1. Restriction Endonuclease(RE): These are the enzyme which cleaves the DNA from

particular sequence. The sequence from where it cleaves the DNA is called

as recognition sequence. Recognition site can be 4 to 8 bp long.

It breaks the nucleotide bond of base pair.

11

TYPES OF RESTRICTION ENDONUCLEASE

• Type I : Made up of three non-identical subunit. Require ATP, mg2+ for activation. They cleave the DNA 1000 bp away from the recognition.

• Type II : Require only mg2+. Made of two identical subunit. Cleaves DNA from recognition site. These are widely used enzyme. More than 300 enzyme are discovered.

• Type III : Cleave 26 bp away from recognition site.

12

CLEAVAGE TYPE

Blunt end ->

Sticky end ->

13

EXAMPLE OF RESTRICTION ENDONUCLEASE

Restriction endonuclease

Source(organism) Recognition sequence

AluI Arthrobacter luteus AG/CTTC/GA

BamHI Bacillus amyloliquefaciens

G/GATCCCCTAG/G

EcoRI Escherichia coli G/AATTCCTTAA/G

HindII H. influenza A/AGCTTTTCGA/A

TaqI Thermus aquaticus T/CGAAGC/T

14

2) Gene library

The gene of interest (DNA fragment) is stored in gene library. There are two gene library available.Genomic library : A collection clones contain all DNA segments of the genome of an organism is called Genomic library.cDNA library : A collection of clones each of which carries a cDNA of an organism is called cDNA.

15

3) VECTOR

A vector is a DNA molecule that has the ability to replicate autonomously in an host cell and into which the DNA fragment to be cloned.Any extra chromosomal small genome/DNA, self replicatinge.G :- Plasmid(pBR322, pUC18/19), Phage(λ phage, phage M13), Cosmid, Phasmid, BAC, YAC.

16

PROPERTIES OF GOOD VECTOR

• Should have origin of replication.

• Should be less than 10kb in size.

• Easy to isolate and purify.

• Easily introduced to host cell.

• Should contain unique target site for many RE.

17

4) HOST CELL

Host cell are the organism in which rDNA are to be transformed. E.g:- The best example for host cell is E. coli.

Properties of good vector:1. Easy to transform.2. Support the replication of rDNA3. Lack active restriction enzyme

18

GENE TRANSFER METHOD

The transfer of rDNA into a bacterial cell or plant cell or animal cell is called gene transfer.

The host cell contain an rDNA is known as transformed cell or recombinant.

The rDNA in cell replicates independently of the chromosomal DNA of cell.

The desired foreign gene present in the rDNA express its characters in host cell.

19

METHODS

In prokaryotes• Transformation• Transduction• Electroporation In eukaryotes• Tranfection• Electroporation • Ultrasonication• Bombardment• Microinjection

20

In vitro PRODUCTION OF INSULIN

• Isolation of gene responsible for insulin production

• Isolation of plasmid• Gene insertion in

plasmid• Introduction of rDNA in

host• Multiplication of

recombinant host cell.

21

TRANSGENIC PLANT

The plant that have been genetically engineered.

They are :-• Herbicide resistance• Insect resistance• Virus resistance• Improved storage • Altered flower color• Environmental stress

resistance• Improved nutritional

quality

22

E.g. BT COTTON

• Bt cotton is genetically modified organism (GMO)cotton variety, which produce insecticide to bollworm.

23

TRANSGENIC ANIMAL

Genetically manipulated animal having an introduced gene are called transgenic animal.

Transgenic mice, sheep, cattle, goats, pigs, poultry and fishes have been developed by using GE.

24

E.g. GLOFISH

The glow fish is a genetically modified fluorescent fish.

Done by bioluminescence, which are found in fireflies or lightning bugs.

A enzyme called as luciferase is taken from firefly and is inserted in fish which make them to glow.

25

APPLICATION• Agriculture Improved crops High yield Resistant High nutritional value Long storage• Medicine

Production of insulin and human growth hormone• Animal husbandry

High milk production High yield of wool

26

Thank you…