Genetic Engineering

• Genetic engineering primarily involves the

manipulation of genetic material (DNA) to achieve
the desired goal in a pre-determined way.
• i. Gene manipulation
• ii. Recombinant DNA (rDNA) technology
• iii. Gene cloning (molecular cloning)
• iv. Genetic modifications
• v. New genetic
 Outline of recombinant DNA technology:

• There are many diverse and complex

techniques involved in gene manipulation.
• Basic principles of recombinant DNA
technology are reasonably simple, and broadly
involve the following stages:
• 1. Generation of DNA fragments and selection of the
desired piece of DNA (e.g. a human gene).
• 2. Insertion of the selected DNA into a cloning vector
(e.g. a plasmid) to create a recombinant DNA or chimeric
DNA
• 3. Introduction of the recombinant vectors into host cells
(e.g. bacteria).
• 4. Multiplication and selection of clones containing the
recombinant molecules.
• 5. Expression of the gene to produce the desired product
• 1. Molecular tools of genetic engineering.
• 2. Host cells-the factories of cloning.
• 3. Vectors-the cloning vehicles.
• 4. Methods of gene transfer.
• 5. Gene cloning strategies.
• 6. Genetic engineering guidelines.
• 7. The future of genetic engineering
 Molecular Tools
• Restriction Endonucleases— DNA Cutting Enzymes:
• Enzymes for the manipulation of DNA.
• Bacterial enzymes that can cut/split DNA (from any
source) at specific sites.
• They were first discovered in E.coli restricting the
replication of bacteriophages, by cutting the viral
DNA
• Enzymes that restrict the viral replication are known
as restriction enzymes or restriction endonucleases.
 Nomenclature:

• Commercially available
• EcoRI - Escherichia (E) coli (co), strain Ry13 (R)
and first endonuclease (I).
• Hindlll
 Types

• 4 different types of restriction endonucleases

are known
• Type 1 (e.g Ecok12)
• Type II (e.g. EcoRI)
• Type III (e.g. EcoPI)
• Type IIs.
• Among these, type II restriction endonucleases
are most commonly used in gene cloning
• The restriction enzymes cleave a DNA to generate a nick
with a 5′ phosphoryl and 3′ OH termini.
• The broken nucleotides form a DNA duplex and exhibit two
fold symmetry from a point.
• In some cases cleavage in two strands are staggered to
produce single strand short projections opposite to each
other with blunt ends of mutually cohesive stickily ends
which are identical and complementary sequences are
called palindrome sequences or palindromes.
• Therefore, when read from 5’→3′, both strands have the
same sequence
 DNA Ligases —DNA Joining Enzymes

• The cut DNA fragments are covalently joined

together by DNA ligases.
• These enzymes were originally isolated from
viruses.
• They also occur in E.coli and eukaryotic cells.
• DNA ligases actively participated in cellular
DNA repair process.
• Permanently hold DNA pieces.
• Hydrogen bonds - are not strong enough
• DNA ligase joins (seals) the DNA fragments by
forming a phosphodiester bond between the
phosphate group of 5′-carbon of one
deoxyribose with the hydroxyl group 3′-carbon
of another deoxyribose
• Phage T4 DNA ligase requires ATP as a cofactor
• E.coli DNA ligase is dependent on NAD+.
• AMP complex that brings about the formation
of phosphodiester bond.
• Results in formation of a recombinant DNA
molecule.
 Homo-polymer tailing:

• Annealing
• Addition of oligo (dA) to 3′-ends of some DNA
molecules and the addition of oligo (dT) to 3′-
ends of other molecules.
• The homo-polymer extensions (by adding 10-40
residues) can be synthesized by using terminal
deoxynucleotidyltransferase (of calf thymus).
• Homo-polymer tailing, achieved by annealing
 Linkers, Adaptors

• Chemically synthesized, short, double-

stranded DNA molecules
• Linkers possess restriction enzyme cleavage
sites
• Ligated to blunt ends
• Adaptors
• Sticky or cohesive ends
Host Cells
 Prokaryotic Hosts

• Escherichia coli:
• organism used in the DNA technology.
• Undoubtedly, E.coli, the simplest Gram
negative bacterium (a common bacterium of
human and animal intestine), has played a key
role in the development of present day
biotechnology
• E.coli can double in number every 20 minutes.
• Plasmids (along with foreign DNA) also
multiply to produce millions of copies, -
• Colony or in short clone.
• Clone is broadly used to a mass of cells,
organisms or genes that are produced by
multiplication of a single cell, organism or
gene.
 Bacillus subtilis:

• Bacillus subtilis is a rod shaped non-

pathogenic bacterium.
• Production of enzymes, antibiotics,
insecticides etc.
• B.subtilis as an alternative to E.coli.
 Eukaryotic Hosts:

• Produce human proteins - to synthesize

complex proteins.
• The most commonly used eukaryotic organism
is the yeast, Saccharomyces cerevisiae.
• Non -pathogenic organism- brewing and baking
industry.
• Certain fungi have also been used in gene
cloning experiments.
 Mammalian cells:

• Certain complex proteins - produced by

mammalian cells
• e.g. tissue plasminogen activator.
• Machinery to modify the protein to its final
form (post-translational modifications).
• Gene manipulation -create transgenic animals
and transgenic plants rather than to isolate
genes for producing specific proteins
 Vectors —The Cloning Vehicles

• Carry a foreign DNA fragment to be cloned.

• Self - replicating in an appropriate host cell.
• The most important vectors are plasmids,
bacteriophages, cosmids and phasmids.
 Plasmids

• extra chromosomal , double- stranded,

circular, self-replicating DNA molecules.
• low copy number (1-4 per cell) / high copy
number (10-100 per cell).
• The size of the plasmids varies from 1 to 500
kb.
• Usually, plasmids contribute to about 0.5 to
5.0% of the total DNA of bacteria
• they carry a set of transfer genes (tra genes)
• copy number
 pBR322
• DNA sequence of 4,361 bp
• resistance for ampicillin (Ampr) and
tetracycline (TeIr) – Markers
• unique recognition sites for RE - EcoRI, Hindlll,
BamHI, Sail and Pstll
• pUC19 (2,686 bp, with ampicillin resistance
gene), and derivatives of pBR322- pBR325,
pBR328 and pBR329.
 Bacteriophages

• Viruses
• DNA gets incorporated into the bacterial
chromosome and remains there permanently.
• Phage vectors can accept short fragments of
foreign DNA into their genomes.
• Hence phage vectors are preferred for working
with genomes of human cells.
• Phage λ consists of a head and a tail (both
being proteins) and its shape is comparable to
a miniature hypodermic syringe.
• The DNA, located in the head, is a linear
molecule of about 50 kb.
• At each end of the DNA, there are single-
stranded extensions of 12 base length each,
which have cohesive (cos) ends.
• The phage DNA has two fates
• Lytic cycle
• Lysogenic cycle.
 Bioreactors

• Continuous Stirred Tank Bioreactors:

• A continuous stirred tank bioreactor consists
of a cylindrical vessel with motor driven
central shaft that supports one or more
agitators (impellers).
• The shaft is fitted at the bottom of the
bioreactor
• The number of impellers is variable
• Ai r is added to the culture medium under pressure
through a device called sparger.
• The sparger along with impellers (agitators) enables
better gas distribution system throughout the vessel.
• The bubbles generated by sparger are broken down to
smaller ones by impellers and dispersed throughout
the medium.
• This enables the creation of a uniform and
homogeneous environment throughout the
bioreactor.
 Bubble Column Bioreactors

• Air or gas is introduced at the base of the column

through perforated pipes or plates, or metal
micro porous spargers
• The flow rate of the air/gas influences the
performance factors — O2 transfer, mixing.
• Perforated plates to improve performance.
• The vessel used for bubble column bioreactors is
usually cylindrical with an aspect ratio of 4-6 (i.e.,
height to diameter ratio).
 Methods of Gene Transfer

• Electroporation:
• Electroporation is based on the principle that high
voltage electric pulses can induce cell plasma
membranes to fuse.
• Electric field-mediated membrane permeabilization.
• Electric shocks can also induce cellular uptake of
exogenous DNA (believed to be via the pores formed
by electric pulses) from the suspending solution.
• Simple and rapid technique for introducing
genes into the cells from various organisms
(microorganisms, plants and animals).
• The cells are placed in a solution containing
DNA and subjected to electrical shocks to
cause holes in the membranes.
• The foreign DNA fragments enter through the
holes into the cytoplasm and then to nucleus.
 Liposome-Mediated Gene Transfer

• Liposomes are circular lipid molecules, which

have an aqueous interior that can carry
nucleic acids.
• Encapsulate DNA in liposomes.
• The liposome- mediated gene transfer,
referred to as lipofection.