BIOTECHNOLOGY: PRINCIPLES

AND PROCESSES

K C MEENA
PRINCIPAL KV JAMALPUR
History of Biotechnology
 1590: Janssen invents the microscope.
 1675:. one of the pioneers of microscopy who in the late 17th
century Leeuwenhoek discovers bacteria and protozoabecame the
first man to make and use a real microscope.
1833: The nucleus of the cell is discovered
 1855: The E. coli bacterium is discovered
 1906: The term "genetics" is used
 1919: The word "biotechnology" is first used
 1938: The term "molecular biology" is used
 1941: The term "genetic engineering" is first used
1980: The U.S. patent for gene cloning is awarded
to Boyer and Cohen.

1981: The first genetically-engineered plant is

reported (TOBACCO)

1981: 1st mice to be successfully cloned

1984: The DNA fingerprinting technique is

developed.
1984: The first genetically-engineered
vaccine is developed against Hepatitis B.

1999: The genetic code of the human chromosome

is deciphered

2001: The sequence of the human genome is published

in Science and Nature
2003: Dolly, the cloned sheep from 1997, is
euthanized
 The term "biotechnology" was coined in 1919 by Karl Ereky, an
Hungarian engineer.
 Definition: the term "biotechnology" refers to the use of living organisms
or their products to modify human health and the human environment.
 Louis Pasteur (1822-1895), the French chemist and microbiologist, is
considered the actual “Father of biotechnology” by most renowned
scientists of the world
 Karoly Ereky (1878-1952) from Hungary coined the term
“biotechnologie” in 1919. At that time, he was food minister in Hungary.
 Daniel Nathans (1928-1999), Nobel laureate in medicine-1978; is
considered “Father of Modern Biotechnology” by many researchers
 Some people consider Kiran Mazumdar Shaw (born in 1953) as father/mother of
biotechnology in India.
 Dr. Pushpa Bhargava (born in 1928); established CCMB (Centre for Cellular and
Molecular Biology) in Hyderabad, in 1977. He is also called “Father of Modern
Biotechnology”.
BIOTECHNOLOGY: PRINCIPLES AND PROCESSES

Two core techniques that enabled birth of modern

biotechnology:

Genetic engineering: Techniques to alter the chemistry of

genetic material (DNA and RNA) to introduce into host
organisms and thus change the phenotype of the host
organism.

Maintenance of sterile (microbial contamination-free)

ambient chemical engineering processes to enable growth of
only the desired microbe/eukaryotic cell in large quantities
RESTRICTION ENZYME VIDEO
Conceptual development of the principle of
genetic engineering
 Asexual reproduction preserves the genetic identity of species.
 Sexual reproduction creates variation and creates unique
combinations of genetic makeup.
 Traditional hybridization procedures used in plant and animal
breeding leads to undesirable genes along with desired genes.
 The techniques of genetic engineering which includes creation
of recombinant DNA, use of gene cloning and gene transfer,
overcome this limitation and allows us to isolate and introduce
only one or a set of desirable genes without introducing
undesirable genes into target organism
 Three basic steps in genetically modifying an organism –
Identification of DNA with desirable gene
Introduction of the identified DNA into the host.
Maintenance of introduced DNA in the host and transfer of the
DNA to its progeny.
TOOLS OF RECOMBINANT DNA
TECHNOLOGY
Restriction Enzymes
Cloning vectors
Competent Host (for transformation with
recombinant DNA)
Before starting with the entire process I would like
to show you a short video clip.
https://www.youtube.com/watch?v=6UiKZKFHb
MQ
 Restriction Enzymes
In the year 1963 two enzymes discovered from Escherichia
coli which restrict the growth of bacteriophage in it.
One of these added methyl groups to DNA.
Other cut the phage DNA. (restriction endonuclease)

The first restriction endonuclease discovered is Hind II.
Hind II always cut DNA molecule at particular point by
recognizing a specific sequence of six base pairs. This is
called recognition sequence for Hind II.
Till date around 900 restriction enzymes isolated from 200
strains of bacteria each of which recognize different
recognition sequences.
 Restriction enzyme belongs to nucleases.
 There are two kind of nucleases:
Exonuclease
Endonuclease

 Exonuclease removes nucleotides from the free ends of the DNA.
 Endonucleases make cuts at specific positions within the DNA.
 Each restriction endonuclease recognizes a specific palindromic
nucleotide sequences in the DNA.
 Palindromes are the group of letters that read same both forward and
backward, e.g. “MALAYALAM”.
 The palindrome in DNA is a sequence of base pairs that reads same
on the two strands when orientation of reading is kept same.

The restriction enzyme cut the strand of DNA little away
from the centre of the palindrome sites, but between the
same two bases on the opposite strand. This leaves single
stranded portions at the ends. There are overhanging
stretches called sticky ends on each strand.
This stickiness of the ends facilitates the action of the
enzyme DNA ligases.
The foreign DNA and the host DNA cut by the same
restriction endonuclease, the resultant DNA fragments
have the same kind of ‘sticky-ends’ and these can be
joined together using DNA ligases.
Convention for naming restriction
endonuclease

The first letter of the name comes from the genus.

Second two letters come from the species of the
prokaryotic cell from which the enzyme isolated
The fourth letter is in capital form derived from the
Strain of microbes.
The Roman letter followed is the order of discovery
Best example: EcoRI comes from Escherichia coli
RY 13
 Separation and isolation of DNA fragments
 The cutting of DNA by restriction endonucleases
results in the fragments of DNA.
 These fragments are separated by a technique
called gel electrophoresis.
 Since the DNA fragments are negatively charged,
they can be separated by forcing them to move
towardsanode under an electric field through
a medium/matrix.
 Most commonly used matrix is agarose, a natural
polymer extracted from sea weed.
 DNA fragments separate according to their size
through sieving effect provided by the agarose gel.
Hence the smaller the fragment size, farther it
moves.
 The separated fragments are visualized by staining
them with Ethidium bromide followed by
exposure to UV radiation.
 The separated bands of DNA are cut out from the
agarose gel and extracted from the gel piece. This
 Cloning vectors:
 The plasmid and bacteriophages have the
ability to replicate within bacterial cells
independent of the control of
chromosomal DNA.
 Alien DNA linked with the vector
multiply its number equal to the copy
number of the plasmid or bacteriophage.
Features of cloning vector:
Origin of replication:
 This is the sequence where the
replication starts called ori gene.
 The alien DNA linked with vector also
replicates.
 Controls the copy number of the linked
DNA.
Selectable marker
It is required to identify recombinant from the non-
recombinant.
Helps in identifying and eliminating non-transformants
and selectively permitting the growth of the transformants.
Transformation is a procedure through which a piece of
foreign DNA is introduced in a host bacterium.
Normally, the gene coding resistance to antibiotics such as
ampicilin. Tetracycline, chloramphenicol or kanamycins
etc are considered as useful selectable markers for E.coli.
The normal E.coli cells do not carry resistance against any
of antibiotics.
Cloning sites
 In order to link the alien DNA, the vector needs to have very few, preferably
single, recognition sites (palindromic site)for the commonly used restriction
endonuclease.
 Commonly used vector is pBR322, for E.coli.
 The ligation of foreign DNA is carried out at a restriction site present in one of
the two antibiotic resistance genes.
 If a foreign DNA ligated or inserted at the Bam H I site of tetracycline
resistance gene in the vector pBR322, the recombinant plasmid will lose
tetracycline resistance. (insertional inactivation)

 The recombinant can be identified from the non-recombinant in following
steps:
 All are grown in ampicilin medium
 One replica of above plate grown in ampicilin medium (control)
 Other replica grown in the medium containing both tetracycline and ampicilin.
 The colonies grows in plate-I but failed to grow in plate-II are identified
as recombinants
Alternative selectable marker
 In E.coli a plasmid called PUK-18 is used as selectable marker,
which is better than pBR322.
 The foreign DNA is introduced within the coding sequence of an
enzyme β-galactosidase, which convert X-Gal (chromatogenic
substrate) into Galactose and 5-bromo+4 chloro indigo (blue
color)
 The non-recombinant produce enzyme and give blue colored
colonies.
 The recombinant unable to produce β-galactosidase and does not
produce blue colored colonies after addition of chromatogenic
substrate i.e. X-Gal.
 This inactivation of insertion of foreign DNA called insertional
inactivation
 Vectors for cloning genes in plants and
animals
 Agrobacterium tumefaciens, a pathogenic bacterium of several dicot
plants.
 This bacterium contains a plasmid called Ti-plasmid.(tumor inducing)
 In natural condition the A.tumifaciens transfer the T-DNA into the plant
which transform normal plant cells into a tumor and direct these tumor
cells to produce the chemical required by the pathogen.
 Retroviruses in animals have the ability to transform normal cells
into cancerous cells.
 The dis-armed retroviruses are being used to transfer gene into animals.
 In Ti-plasmid the T-DNA is replaced by the gene of interest, still
A.tumifaciens able to transfer the gene into the plant without causing
tumor in plants.
Competent Host (for transformation with recombinant DNA)
 DNA is a hydrophilic molecule; it cannot pass through cell membranes.
 In order to force bacteria to take-up the plasmid, the bacterial cells must
first be made ‘competent’ to take up DNA.
 The bacterial cell is treated with divalent cations such as calcium, which
increases the efficiency of DNA up take by the bacteria.
 Recombinant DNA and the bacterial cells are incubated in ice, followed
by placing them briefly at 42oC (heat shock) and then putting them back
in ice.
 By microinjection the recombinant DNA directly injected into the
nucleus of the animal cell.
 Plant cells are bombarded with high velocity micro-particles of gold or
tungsten coated with DNA in a method known as biolistics or gene gun.
 The disarmed pathogen vectors which when allowed infecting the cell
transfer the recombinant DNA into the host.
PROCESS OF RECOMBINANT
TECHNOLOGY:
Isolation of DNA ,
Fragmentation of DNA by restriction endonuclease.
Isolation of desired DNA fragment by gel
electrophoresis.
Ligation of DNA fragment with a vector by DNA ligase
Transferring the recombinant DNA into the host
Culturing the host cells in a medium at large scale in
a bioreactor.
Extraction of desired product
by downstream processing.
Isolation of the Genetic material (DNA):
Bacterial cell wall digested by Lysozyme.
Plant cell wall is digested by cellulase and pectinase.
Fungal cell wall is digested by chitinase.
RNA of the cellular content is digested
by ribonuclease.
Proteins are removed by Proteases.
Purified DNA ultimately precipitated out after addition
of chilled ethanol.
The precipitated DNA is separated and removed
by spooling.
Amplification of Gene of Interest using PCR
PCR stands for Polymerase chain reaction:
Multiple copy of gene of interest can be synthesized in
vitro.
PCR includes following steps:
Denaturation
Double stranded DNA made single stranded.
It is done by heating the DNA at 94oC.
Each single stranded DNA is called Template strand
Annealing
 Two sets of primer (small oligonucleotide chain that are
complementary to the DNA at 3’ end of the DNA template) added
to the medium.
 This is done at around 50oC.
Extension
 Deoxyribonucleotides triphosphates are added in the medium.
 Taq polymerase catalyses the polymerization reaction using
nucleotides extending from the primer towards 5’ end of the
template.
 Taq polymerase is a thermostable polymerase isolated from a
bacterium called Thermus aquaticus.
 It catalyses polymerization reaction at 74 oC.
Obtaining the Foreign Gene product or
Recombinant product:
The protein encoding gene is expressed in
a heterogeneous host is called a recombinant
protein.
The host is cultured in a continuous culture system
provided in bioreactor.
A bioreactor provides optimum growth conditions
(temperature, pH, substrate, salts, vitamins, oxygen)
Bioreactor covert the raw materials into specific
product, specific enzyme.
Downstream processing
After biosynthesis inside the bioreactor, the product
has to be subjected through a series of processes before
it is ready for marketing.
The process includes separation and purification,
which are collectively referred as downstream
processing.
The product has to be formulated in suitable
preservatives.
Such formulation has to undergo through clinical trials
as in case of drugs.