BIOTECHNOLOGY: PRINCIPLES

AND PROCESSES

Nithina M
PGT BIOLOGY
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
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 lead to inclusion of undesirable
genes along with desired genes.
 The techniques of genetic engineering which includes
creation
of recombinant DNA, use of gene cloningand
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
TOOLS OF RECOMBINANT DNA
TECHNOLOGY
Restriction Enzymes
Cloning vectors
Competent Host (for
transformation with
recombinant DNA)
 Restriction
Enzymes
In the year 1963 two enzymes discovered
from Escherichia coli which restrict the
growth of bacteriophagein 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
 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 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
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
 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
 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
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.

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.
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
 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
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
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.
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
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
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
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,
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.