GENETIC ENGINERING

Kishor Umesh KamatagiPALB 4066Dept. of Plant Pathology UAS, Bengaluru

What is genetic engineering

• Genetic engineering, also known as recombinant DNA technology, means altering the genes in a living organism to produce a Genetically Modified Organism (GMO) with a new genotype.

• Term “Genetic Engineering” was coined by Jack Williamson.

• In 1972, Paul berg created the first recombinant DNA molecules by combining DNA from the monkey virus SV40 with that of the lambda virus.

• In 1973 Herbert Boyer and Stanley Cohen created the first transgenic organism by inserting antibiotic resistance genes into the plasmid of an E. coli bacterium.

• The first trials of genetically engineered plants occurred in France and the USA in 1986, tobacco plants were engineered to be resistant to herbicides.

• The Flavr Savr tomato was a tomato engineered to have a longer shelf life. In 1995, Bt Potato was approved safe by the Environmental Protection Agency.

• Bt-Cotton is a genetically modified cotton which is resistant to pests. Golden Rice genetically modified to contain beta-carotene(a source of Vitamin A).

Enzymes: Restriction Endonucleases

• Enzymes that cut DNA• Each has a known

sequence of 4 to 10 pairs as its target

• Can recognize and clip at palindromes– Madam, I’m Adam– GAATTC– CTTAAG

Restriction endonucleasemakes staggered cutat palindrome.

(1)

(2)

Sticky ends

Action of restriction endonucleases. (1) A restriction endonucleaserecognizes and cleaves DNA at the site of a specific palindromic sequence. Cleavage can produce staggered tails called sticky ends that accept complementary tails for gene splicing. (2) The sticky ends can be used to join DNA from different organisms by cutting it with the same restriction enzyme, ensuring that all fragments have complementry ends.

(c)

DNAOrganism

2

DNAOrganism

1

Site of cut

G A T C

C T A G

G A T CC T A G

TGC

A

ACGT

TGCA

ACGT

TGC

A

ACGTTGCA

ACGT

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Some Common Restriction Enzymes

• BamHI - Bacillus amyloliquefaciens G|GATCC

CCTAG|G

• EcoRI - Escherichia coliG|AATTC

CTTAA|G

• EcoRV - Escherichia coliGAT|ATC

CTA|TAG

Enzymes: Ligase and Reverse Transcriptase

– Ligase: Enzyme necessary to seal sticky ends together.

– Reverse transcriptase: enzyme that is used when converting RNA into DNA.• Copied DNA is referred to as complementary DNA or cDNA

Vectors

Vectors are autonomous replicating DNA molecule it may be natural one (plasmids & Bacteriophage) or artificial once (cosmids, YAC & BAC) used to carry desired gene from donor cell to host cell for its cloning is called vector.

Properties of a good vector

• It should be able to replicate autonomously.• A vector should be ideally less than 10 kb in size.• Vector should be easy to isolate & purify.• It should be easily introduced into the host cells.• The vector should have suitable marker genes.• Vector should contain multiple cloning site.• It should have the ability to integrate either itself or the

DNA insert it carries into the genome of the host cell.• It should contain at least suitable control elements viz

promoter, operator & ribosome binding sites.

Properties of a Good Host

• Should be easy to transform• Should support the replication of rDNA • Should be free from elements that interfere replication of

rDNA • Lack the active restriction enzyme Eg. E. coli strain HB 101.• Should not have methylase enzyme that methylate the

DNA, that become resistant to use full restriction enzymes.• Should deficient in normal recombination function, so that

DNA insert is not altered by recombination.

Types of vectors

Cloning vector 1. Plasmid vector2. Bacteriophage vector3. Cosmid vector4. Phagemid vector 5. Phasmid vector6. Artificial chromosomes

Expression vector

Cloning vectorA vector used for transfer and multiplication of desired DNA in

suitable host is called as cloning vector.

Plasmid: The extra chromosomal, self replicating, double stranded & circular DNA molecules of bacteria that carries the characters like sex factor, drug resistance, colicin factor etc. is called as Plasmid.

Cosmid are essentially plasmids that contain minimum of 250 bp of lambda DNA.

Phagemid: A plasmid vector that contains origin of replication from a phage, in addition to that of plasmid.

Phasmid: a gene-cloning vector consisting of an artificial combination of a plasmid with a phage such that its genome contains functional origins of replication of both; it may thus be propagated either as a plasmid or as a phage in appropriate host strains.

Expression vector

Process of Genetic Engineering

Five steps involved in this process:1. Isolation

2. Cutting

3. Insertion (Ligation)

4. Transformation

5. Expression

Step 1: Isolating the gene

• Proteins called “restriction enzymes” are used to cut the DNA into small pieces.

Step 2: Inserting gene into vector

• Vector – molecule of DNA which is used to carry a foreign gene into a host cell.

Step 3: Cloning Vector

• All genetically identical because of asexual reproduction

Outcomes of Genetic Engineering

• Researches have succeeded in disrupting the tumor causing genes and inserting new genes for resistance to disease, frost and herbicides.

Basic steps in transformation of plant cells by Agrobacterium tumefaciens.

• The Ti plasmid can be removed from the agrobacterium, cut open with a restriction endonuclease and mixed with DNA from another source (foreign DNA) that has been digested with the same restriction enzyme.

• The foreign DNA is then inserted into the plasmid

The plasmid can then be put back into agrobacterium

by transformation. Agrobacterium attaches to the plant cell and a copy of the

plasmid is transferred into the plant cell.

• The T-DNA carrying useful information then integrates

into the plant DNA

• When a plant cell divides, each daughter cell receives

the new gene.• In some cases the entire plant with the new trait can be

generated from a single cell.

Disadvantages of GE

• Modified genes can have an irreversible effects and associated consequences.

• Unknown Consequences of Viral Genes• Uncertain Effects that may be brought by genetically

modified life form.

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