Chapter

Biotechnology
By:-
Dr.Zeeshan Ahmed
aspirationclasses@i
cici
• BIOTECHNOLOGY

• Biotechnology can be defined as the use of microorganisms, plants or animal

cells or their components to produce products and processes useful to
humans.
• According to the European Federation of Biotechnology (EFB), biotechnology
is the integration of natural science and organisms, cells, parts thereof and
molecular analogues for product’s and services. The term 'Biotechnology' was
coined by Karl Ereky in 1919.
• 2. Principles of biotechnology are based on the concept of the following
techniques:
• (i) Genetic engineering is the technique to alter the chemistry of genetic
material(DNA/RNA), to introduce these into another organisms and thus,
change the phenotype of the host organism.
• (ii) Adequate maintenance of sterile conditions to support growth of only the
desired microbes /eukaryotic cells in large quantities for the manufacture of
biotechnological products like antibiotics, vaccines, enzymes, etc.
• The techniques of genetic engineering include the following:
• (i) Creation of recombinant DNA by combining desired genes.
• ii) Gene transfer.
• (iii) Maintenance of DNA in host and gene cloning.
• The basic steps in genetic engineering can be summarised as:
• (i) Identification of DNA with desirable genes.
• (ii) Introduction of the identified DNA into the host.
• (iii) Maintenance of introduced DNA in the host and transfer of the DNA to
its progeny .
• At last recombinant protein is obtained from host through a process
known as Down Stream Processing (DSP).
• Construction of First Artificial Recombinant DNA
• i) It was achieved by linking a gene encoding antibiotic resistance with a
native plasmid (an autonomously replicating circular extrachromosomal
DNA) of Salmonella typhimurium.
• (ii) Stanley Cohen and Herbert Boyer accomplished this in 1972.
• (iii) They isolated the antibiotic resistance gene by cutting out a piece of
DNA from a plasmids.
• (iv) The cutting of DNA at specific locations was carried out by molecular
scissors i.e. restriction enzymes.
• (v) The cut piece of DNA was then linked to the plasmid DNA with the
enzyme DNA ligase. The plasmid DNA acts as vectors to transfer the
piece of DNA attached to it.
• (vi) When this DNA is transferred into E. coli, it could replicate using the
new host’s DNA polymerase enzyme and make multiple copies.
• (vii) This ability o multiply copies of antibiotic resistance gene in E. coli
was called cloning of antibiotic resistance gene in E. coli.
• Tools of recombinant DNA technology are as follow:-
• (i) Restriction enzymes. (iv) Vector
• (ii) Polymerase enzymes. (v) component host organisms
• (iii) Ligases
• (i) Two enzymes from E. coli that were responsible for restricting the
growth of bacteriophage were isolated in 1963, one of them added methyl
group to DNA and the other cut DNA into segments. The later was called
restriction endonuclease.
• (ii) The first restriction endonuclease Hind II was isolated by Smith Wilcox
and Kelley(1968). They found that it always cut DNA molecules at a
particular point by recognising a specific sequence of six base pairs known
as recognition sequence.
• (iii) Besides Hind II, more than 900 restriction enzymes have been isolated
now, from over 230 strains of bacteria, each of which recognise different
recognition sequences.
• Naming of Restriction Enzymes
• (a) The first letter is derived from the genus name and the next two
letters from the species name of the prokaryotic cell from which
enzymes are extracted.
• (b) The Roman numbers after name show the order in which the
enzymes were isolated from the bacterial strain.For example, Eco RI
comes from Escherichia coli RY13 and Eco RII comes from E. coli R 245,
etc.
• Restriction enzymes belong to a class of enzymes called nucleases.
• Nucleases are of two types:
• Exonucleases -They remove nucleotides from the ends
• Endonucleases -They cut at specific positions within the DNA. These
specific sequences are recognition sequences.
• (a) Each restriction endonuclease recognises a specific palindromic
nucleotide sequences in the DNA.
• (b) Palindrome in DNA is a sequence of base pairs that reads same on the
two strands when orientation of reading is kept same.
• For example, the following sequences reads the same on the two strands
in
• 5— 3 direction as well as 3’— 5 direction.
• MECHANISM OF ACTION OF RESTRICTION ENZYMES
• (a) Restriction enzymes cut the strand of DNA a little away from the
centre of the PALINDROME sites, but between the same two bases on the
opposite strands.
• (b) This leaves single-stranded portions at the ends.
• (c) There are overhanging stretches called sticky ends on each strands as
given in above figure. These are named so, because they form hydrogen
bonds with their complementary cut counterparts.
• (d) The stickiness of the ends facilitates the action of the enzyme DNA
ligase.
• (e) Restriction endonucleases are used in genetic engineering to form
recombinant molecules of DNA, which are composed of DNA from
different sources/genomes.
• (f) These sticky ends are complementary to each other when cut by same
restriction enzyme, therefore can be joined together (end-to-end) using
DNA ligases.
• Separation and isolation of DNA fragments
• The cutting of DNA by restriction endonucleases results in the fragments
of DNA.
• These DNA fragments can be separated by a technique called Gel
electrophoresis
• DNA fragments are negatively charged. They can be separated by forcing
them to move towards the anode under an electric field through a
medium/matrix.
• Most commonly used matrix is agarose. Agarose is a natural polymer
extracted from sea weeds.
• The DNA fragments separate (resolve) according to their size through
sieving effects provided by the agarose gel.Hence, the smaller sized
fragment move farther.
• Ethidium bromide is used as stain for DNA, which on exposure to UV-light
appear as orange coloured bands.
• Separated bands of DNA are cut out from agarose gel.This is called elution.
• These DNA fragments are used in recombinant DNA by joining them with
cloning vectors.
• DNA Ligase-These enzymes repairs broken DNA by joining two
nucleotides in a DNA strand. It is used in recombinant DNA technology
to join complementary restriction fragments (reverse of restriction
endonuelease ).
• Lyasess-These are the enzymes used to open the cells or dissolve the
cell wall.
• Synthetases-It helps in either in vitro synthesis of DNA or in the
synthesis of complementary DNA.e.g. DNA polymerase.
• Cloning vectors are the DNA molecules that gan carry a foreign DNA
segment into the host cell.
• i) The vectors used in recombinant DNA technology can be:
• (a) Plasmids- Autonomously replicating circular extra-chromosomal DNA.
• (b) Bacteriophages- Viruses infecting bacteria.
• (c)Cosmids- Hybrid vectors derived from plasmids which contain cos site
of A phage.
• (d) Tumour inducing (Ti) plasmid – Agrobacterium tumefaciens is used as
a cloning vector. It is known as natural engineer of plants.
• (e) Retrovirus- These viruses have been modified and are used to carry
desirable genes into animal cells.
• (f) Bacterial Artificial Chromosomes Vectors- These are vectors based on
natural, extra chromosomonal of E.coli.
• (g) Yeast Artificial Chromosomes Vectors- These are vectors obtained by
joining segment of DNA from yeast with the linear plasmid from bacteria.
• Features Required to Facilitate Cloning into Vector
• (a) Origin of replication (Ori)
• (b) Selectable marker
• (c)Cloning sites
• (d) Vectors for cloning genes in plants and animals.
• (a) Origin of replication (Ori) is a sequence from where replication
starts.
• Any piece of DNA when linked to this sequence can be made to
replicate within the host cells.
• The sequence is also responsible for controlling the copy number of
the linked DNA.
• So if one wants to recover many copies of target DNA it should be
cloned in a vector whose origin support high copy number.
• (b) Selectable marker helps in identifying and eliminating non-
transformants and selectively permitting the growth of the
transformants.
• Transformation is a process through which a piece of DNA is
introduced in a host bacterium. Those host cells, which have got
recombinant DNA are known as transformant, while those have not got
the recombinant DNA are known as non-transformant.
• Ligation of alien DNA is carried out at a restriction site present in one of
the two antibiotic resistance genes. Example is ligating a foreign DNA at
theBam HI site of tetracycline resistance gene in the vector pBR322.
• The recombinant plasmids will lose tetracycline resistance due to
insertion of foreign DNA. But, it still can be selected out from non-
recombinant ones by plating the transformants on ampicillin containing
medium.
• The transformants growing on ampicillin containing medium are then
transferred on a medium containing tetracycline.
• The recombinant will grow in ampicillin containing medium but not on that
containing tetracycline. Because in recombinants tetracycline antibiotic
resistance gene is disturbed.
• The non-recombinants will grow on the medium containing both the
antibiotics, Because both antibiotic resistance genes are functional.
• In this example, one antibiotic resistance gene helps in selecting the
transformants whereas, the other antibiotic resistance gene gets
'inactivated, due to insertion' of alien DNA and helps in selection of
recombinants.
• Selection of recombinants due to inactivation of antibiotics is a
cumbersome procedure , because it requires simultaneous plating on two
plates having different antibiotics. So alternative selectable markers are
developed which differentiate recombinants from non-recombinants on
the basis of their ability produce colours in the presence of a chromogenic
substrate.
• In this method, a recombinant DNA is inserted within the coding sequence
of an enzyme B-galactosidase. So if gene of interest gets incorporated B-
galactosidase gene will be disturbed.
• This results into inactivation of the enzyme B-galactosidase (insertional
inactivation ) and B-galactosidase enzyme will not be produce.
• The bacterial colonies whose plasmids do not have an insert ,produce blue
colour, because B-galactosidase enzyme produce a colour, but others do
not produce any colour, when grown on a chromogenic substrate, because
here B-galactosidase enzyme is not produced. This results into inactivation
of enzyme, which is referred to as insertional inactivation.
• (c) cloning sites are required to link the alien DNA with the vector.
• The vector requires very few or single recognition sites for the
commonly used restriction enzymes..
• The presence of more than one recognition sites within the vector will
generate several fragments leading to complication in gene cloning. So
one recognition site for an enzyme is preferred.
• d) Vectors for cloning genes in plants and animals are many which are
used to cione genes in plants and animals.
• (d) Vectors for cloning genes in plants and animals are many which are
used to clone genes in plants and animals.
• In plants, the Tumour inducing (Ti) plasmid of Agrobacterium
tumefaciens is used as a cloning vector. It is known as natural
engineer of plants.
• Agrobacterium tumefaciens is a pathogen of several dicot plants.
• It delivers a piece of DNA known as T-DNA in the Ti plasmid which
transforms normal plant cells into tumour cells to produce chemicals
required by pathogens.
• The tumour inducing plasmid of this bacteria has now been modified
into a cloning vector which is no more pathogenic to the plants.
• Retrovirus, adenovirus, papillomavirus are also now used as cloning
vectors in animals because of their ability to transform normal cells
into cancerous cells.
• Competent host organism (for transformation with recombinant DNA) is
required because DNA being a hydrophilic molecule, cannot pass through
cell membranes.Hence, the bacteria should be made competent to
accept the DNA molecules.
• (1) Competency is the ability of a cell to take up foreign DNA.
• (2) Methods to make a cell competent are as follow:
• (a) Chemical method- In this method, the cell is treated with a specific
concentration of a divalent cation such as calcium to increase pore size in
cell wall.The cells are then incubated with recombinant DNA on ice,
followed by placing them briefly at 42°C and then putting it back on ice.
This is called heat shock treatment . Because of this permeability of
bacterial cell increases. 'This enables the bacteria to take up the
recombinant DNA.
• (b) Physical methods In this method, a recombinant DNA is directly
injected into the nucleus of an animal cell by microinjection method.In
plants, cells are bombarded with high velocity microparticles of gold or
tungsten coated with DNA called as biolistics or gene gun method.
• (c) Disarmed pathogen vectors when allowed to infect the cell, transfer
the recombinantDNA into the host.
• Topic 2 Processes of RecombinantDNA Technology
• Recombinant DNA technology involves the following steps in sequence:-
• 1) Isolation of the genetic material (DNA) is carried out in the following
steps:-
• (a) DNA is enclosed within the membranes. To release DNA along with
other macromolecules such as RNA, proteins, polysaccharides and lipids,
bacteria cell/plant or animal tissue are treated with enzymes such as
lysozyme (bacteria) cellulase (plant cells), chitinase (fungus).
• (b) RNA can be removed by treatment with ribonuclease.
• (c) Proteins can be removed by treatment with protease.
• 2) Cutting of DNA at specific locations is done by using restriction
enzymes .The purified DNA is incubated with the specific restriction
enzyme at conditions optimum for the enzyme to act.
• 3) Isolation of desired DNA fragments is carried out using agarose gel
electrophoresis.
• 4) Amplification of gene of interest using Polymerase Chain Reaction
(PCR) is a reaction in which multiple copies of specific DNA (gene of
interest) sequence are made (amplification) in vitro. The technique was
developed by Kary Mullis in 1985 who received Nobel Prize for chemistry
in 1993.
• (a) PCR technique requires
• A DNA template, which is a double-stranded DNA that needs to be
amplified.
• Primers are small chemically made oligonucleotides of about 10-18
nucleotides that are complementary to a region of template DNA.
• Enzymes used are DNA Taq polymerase (from a bacterium, Thermus
aquaticus).
• Deoxyribonucleotides for synthesis of new strand.
• (b) Steps in PCR
• Denaturation of double-stranded DNA is carried out by applying high
temperature of 95°C for 15 seconds. Each separated single-strand acts
as a template for DNA synthesis.
• Annealing is carried out by two sets of primers, which are added in the
reaction.They anneal to the 3 end of each separated strand. Primers act
as initiator of replication.
• Extension is done by DNA polymerase by adding nucleotides
complementary to the template in the reaction.
• A thermostable DNA polymerase (Taq polymerase) is used in the
reaction, which can tolerate the high temperature of the reaction.
• These steps are repeated many times to obtain several copies of the
desired DNA.
• 5) Ligation of DNA fragment into a vector requires a vector DNA and
source DNA.
• 6) Insertion of recombinant DNA into the host cell/organism .
• 7) Culturing the host cells:- The cell containing the foreign gene is
cultured on an appropriate medium at optimal conditions. The DNA gets
multiplied.
• 8) Extraction of desired gene product is carried out in the following steps:
• (a) A protein encoding gene expressed in a heterologous host is called
recombinants protein.
• (b) Cells having genes of interest can be grown on a small or on a large
scale.
• (c) In small scale, cells are grown on cultures and then expressed protein
is extracted and purified by various separation methods.
• (d) In large scale, cells are grown in a continuous culture system in which
fresh medium is added from one side to maintain cells growth phase and
the desired protein is collected from the other side.
• Bioreactors- are the large volume (100-1000 L) vessels in which raw
materials are biologically converted into specific products, individual
enzymes, etc., using microbial plant, animal or human cells.

• (i) It provides the optimal conditions for achieving the desired product
by providing optimal growth conditions like temperature, pH, substrate,
salt, vitamins and oxygen.
• (ii) The most commonly used bioreactors are of stirring type:
• (a) Simple stirred tank bioreactor
• (b) Sparged stirred-tank bioreactor
• (iii) The stirred-tank reactor is usually cylindrical or with a curved base
to facilitate the mixing of the reactor contents.
• (iv) The stirrer facilitates mixing and oxygen availability throughout the
bioreactor.
• (v)In the sparged stirred-tank bioreactor, sterile air bubbles are sparged,
to increase the surface area for oxygen transfer.
• (vi) The components of a bioreactor are:
• (a) An agitator system (b) An oxygen delivery system
• (c) A foam control system (d) A temperature control system
• (e) pH control system (f) Sampling ports to withdraw small volumes of
culture periodically.
• 3. Downstream processing:- After the product formation in bioreactions,
they are subjected to a series of processes, collectively referred to as
downstream processing.
• These include:- (i) Separation of desired product.
• (ii) Purification of products.
• (ii) Formulated with preservatives.
• Finally, these products undergo strict quality control testing before being
marketed as a finished products.