Biotechnology - Principles and Processes

 The use of organisms, enzymes, plants or animal cells or their components to generate
products and services useful to human beings.
 As per European Federation of Biotechnology (EFB), biotechnology is the integration of
natural science and organisms, cells, parts thereof and molecule analogues for products and
services.
 The term Biotechnology was coined by Hungarian Engineer, Karl Ereky (1917).
• Biotechnology can be studied in two phases:
 Traditional biotechnology (based on the natural capabilities of organisms.
Ex. Wine, beer, cheese, curd, etc…
 Modern biotechnology (processes which use genetically modified organism to
achieve products on larger scale. Ex. Test tube baby, DNA vaccine, insulin
production.
Principles of Biotechnology
The Science of Modern Bio technology is based mainly on two core techniques.
1. Genetic Engineering:
The technique which alters the chemistry of genetic material (DNA and RNA) to introduce these
into host organisms and thus change the phenotype of the host organisms.
Paul Berg is often considered “Father of Genetic Engineering” and was awarded Nobel prize in
1980.
2. Maintenance of Contamination Free (Sterile) conditions:
The technique to facilitate the growth and multiplication of only the desired microbes or cells in
large numbers under sterile conditions for the manufacture of biotechnological products like
antibiotics, vaccines, enzymes, etc…
Conceptual Principles of Genetic Engineering
The technique of genetic engineering involves:
 Creation of recombinant DNA,
 Use of gene cloning
 Gene transfer
PROCESS:
 When a piece of DNA is introduced into alien (foreign) organism, it would not be able to
multiply itself in the progeny cells of the organisms. But when it gets integrated into the
 genome of the recipient, it may multiply and be inherited along with the host DNA. It is
because the alien piece of DNA has become part of a chromosome which has the ability to
replicate. There is a specific DNA sequence in the chromosome called origin of replication
which is responsible for initiating replication. Therefore, for the replication of any alien
piece of DNA, it must join a chromosome which has an origin of replication (ori), so that
this alien piece of DNA can replicate and multiply itself in host organism. This is referred
to as cloning or making identical copies of any template DNA.
Construction of artificial recombinant DNA molecule
The construction of first recombinant DNA emerged from the possibility of linking a gene
encoding antibiotic resistance with a native plasmid of salmonella typhimuriun. Plasmid is self
replicating extra chromosomal circular DNA present in some bacteria.
Two scientists, Stanley Cohan and Herbert Boyer (California university), constructed the first
recombinant DNA in 1972 by following the below steps:
 Isolating antibiotic resistance gene
 Cutting out a piece of DNA from a plasmid which was responsible for giving antibiotic
resistance.
 Cutting of DNA at a specific location is possible by restriction enzymes popularly known
as molecular scissors.
 Link to plasmid - the cut piece of DNA was then linked with a plasmid DNA. These
plasmid DNA acts as vectors to transfer the piece of DNA attached to it.
• The linking of antibiotic resistance gene with the plasmid vector is done with the help of
enzyme DNA ligase. DNA ligase acts on cut DNA molecules and joins their ends. This
makes a new combination of circular autonomously replicating DNA created in vitro, it is
known as recombinant DNA. When this recombinant DNA is transferred into a bacterium,
it could replicate using the new hosts DNA polymerase enzyme and make multiple copies.
The making of multiple copies of antibiotic resistance gene was termed as cloning of
antibiotic resistance gene in E.Coli.
 Example: Female anopheles mosquito acts as an insect vector to transfer the malarial
parasite into human body. The same way, a plasmid can be used as vector to transfer an
alien piece of DNA into the host organism.
 Hence, we can infer that there are three basic steps in genetically modifying an organism –
(i) identification of DNA with desirable genes, (ii) introduction of identified DNA into the
host, (iii) maintenance of introduced DNA in the host and transfer of DNA to its progeny.
Tools of Recombinant DNA Technology:
Restriction Enzymes (The Molecular Scissors)
 Restriction enzymes belongs to large class of enzymes called nucleases. There are two
kinds:
  Exonucleases: Remove nucleotides from the ends of DNA. They don’t cut RNA, act on
single strand of DNA or gaps in double stranded DNA.
 Endonucleases: Makes cut at specific positions within the DNA. They may cut RNA,
cleave one strand or both strands of double stranded DNA.
 There are three classes of restriction enzymes differing slightly in their mode of action.
 Type I
 Type II
 Type III
 Out of these, only type II restriction enzymes are used in recombinant DNA technology
because they can be used in vitro to recognize and cut within specific DNA sequence
typically of 4 to 8 nucleotides. The first restriction endonuclease was Hind II. Its
functioning depended on a specific DNA nucleotide sequences. It always cuts DNA
molecules at a particular point by recognizing a specific sequence of 6 base pairs. This
specific base sequence is known as the recognition sequence for Hind II. Presently, more
than 900 restriction enzymes are known, which have been isolated from over 230 strains of
bacteria.
NOMENCLATURE OF RESTRICTION ENZYMES
 Naming of restriction enzymes is based on the name of bacterium from which they have
been isolated. The first letter of the name comes from the genus, second two letters come
from the species of the bacterium. Next is the strain of the organism, last in a roman
numeral signifying the order in which the enzyme was isolated from the strain of the
bacteria.
 E.g. ECORI comes from Escherichia Coli RY13.
ECORI
Action of Restriction Endonuclease
 Functions by inspecting the length of a DNA sequence. Once it finds its specific
recognition sequence, it will blind to the DNA and cut each of the two strands of the double
helix at specific points in their sugar phosphate backbones.
 Each restriction endonuclease recognizes a specific palindromic nucleotide sequence in the
DNA. The palindrome is a group of letters that reads the same forwards and backwards e.g.
Malayalam.
MALAYALAM
 The palindrome in DNA is a sequence of base pairs that reads same on the two strands
when orientation of reading is kept the same, e.g the following sequences read the same on
the two strands in 5’ 3’ direction. This is also true when we read 3’ 5’
direction.
 5’G AA TT C -3’
 3’C TT AA G -5’ palindromic sequence
 Several restrictions enzymes cut strands of DNA a little away from the centre of the
palindrome sites, but between the same two bases on the opposite strands. This leaves a
single stranded portions at the end. There are overhanging stretches called sticky ends on
each strand. These are named so because they form hydrogen bonds with their
complimentary cut counter parts. This stickiness of the ends helps enzymes DNA ligase in
joining DNA fragments.
 When cut by the same restriction enzyme, the resultant DNA fragments have the same kind
of ‘sticky ends’ and these can be joined together end to end using DNA ligase .
 In Genetic Engineering, restriction endonucleases are used to form recombinant DNA
molecules, composed of DNA from different sources/ genomes.
Action of Restriction Enzyme

Some Other Enzymes Used in Recombinant DNA technology

 Besides restriction enzymes, several other enzymes also pay significant role in recombinant
DNA technology . Some of them are as follows:
 DNA ligase (Molecular glue).
 Alkaline phosphatase (AP).
 DNA polymerase.
Separation and Isolation of DNA Fragments (Gel Electrophoresis)
 When DNA is treated by restriction endonucleases, fragments of DNA are formed. These
fragments are separated by a technique called gel electrophoresis.
 Electrophoresis is a technique of separation of charged molecules under the influence of an
electric –field so that they migrate in the direction of electrode bearing the opposite charges
through a base material called matrix. This technique was developed by A. Tiselius in
1937.
 The base material/ matrix through which molecules travel may be polyacrylamide
(Polyacryline Gel Electrophoresis or PAGE) or Agarose (Agarose Gel Electrophoresis or
AGE).
 Nowadays, Agarose is most commonly used matrix which is a natural polymer extracted
from sea weeds. Agarose dissolved in hot water, which on cooling forms double helices of
thick filaments.
  The latter become cross linked to form the gel. Since DNA fragments are negatively
charged molecules, they can be separated by forcing them to move towards the anode under
an electric field through the matrix.
 The DNA fragments separate according to their size through the pores of agarose gel.
Hence the smaller the fragment size, the farther it moves.
 The DNA fragments can be visualized after staining with ethidium bromide and exposing
to UV radiation.
 Pure DNA cannot be seen in the visible light and without staining.
 DNA fragments appear as bright orange colored bands in the ethidium bromide stained gel
exposed to UV light.
 The separated bands of DNA are cut out from the agarose gel and extracted out from the
gel piece.
 The process of separation and extraction of DNA fragments from two different sources are
used in the formation of recombinant DNA by joining them with help of cloning vectors.

Gene Cloning
 The term ‘clone’ means an exact duplicate copy of an original material.
 Biotechnologist, particularly a genetic engineer, used the term cloning for production of
multiple and identical copies of the gene (DNA) inside the host cell (usually a bacterium).
 This kind of cloning is specifically called Gene cloning.
Cloning Vectors
Cloning vectors (vehicles for cloning) used in genetic engineering includes:
 Plasmid Vectors, Bacteriophages Vectors & Animal And Plant Viruses
  Plasmids and Bacteriophages have the ability to replicate within the bacterial cells,
independent of the control of chromosomal DNA.
 Bacteriophages, because of their high no. per cell, have very high copy numbers of genomes
within the bacterial cells.
 Some plasmids have one or two copies per cell, whereas others have 15-100 copies per cell.
Their number can go even higher.
 If you are able to link an alien piece of DNA with bacteriophage or plasmid DNA, we can
multiple its number equal to the copy number of plasmid or bacteriophage.
Plasmid Vectors
 Plasmids are usually not essential for normal cell growth and division. They often confer
some traits on the host organism, e.g resistance to certain antibiotics or toxins. That can be a
selective advantage under certain conditions. They are small, extra chromosomal, self
replicating, usually circular double stranded DNA molecules that occur naturally in many
bacteria & yeast.
 One of the easily manipulated plasmids used as vectors in PBR322 and it is the first artificial
cloning vector constructed by Boliver and Rodriquez 1977.
Features of Cloning Vector
The features that are required in a vector to facilitate cloning of a gene are as follows:
1. Origin of replication (ORI)
2. Selectable marker
3. Cloning sites (recognition sites)
Origin of replication (ORI)
 The sequence in a DNA molecule from where replication starts is called ORI. Any piece of
DNA when linked to ORI can be made to replicate within host cells.
 This sequence is also responsible for controlling the copy number of the linked DNA.
 Therefore, when many copies of the target DNA are required, it is cloned in a vector whose
origin support high copy number.
Selectable Marker
 The vector also requires a selectable marker. It helps in identifying and eliminating non
transformants by selectively permitting the growth of the transformants, a bacterium which
has modified due to the introduction of a piece of foreign DNA called transformant.
 The process through which a piece of DNA is introduced in a host bacterium is called
transformation.
  Normally, the genes encoding resistance to antibiotic such as ampicillin, choramphenicol,
tetracycline or kanamycin etc are considered useful selectable markers for E.Coli. The
normal E.Coli cells do not carry resistance against any of these antibiotics.
Cloning Sites (Recognition Sites)
 The vectors must have very few preferably single recognition sites for the activity of
commonly used restriction enzymes in order to link the foreign DNA.
 The presence of more than one recognition sites within the vector will produce several
fragments which will make the process of gene cloning more complicated. The ligation of
foreign DNA is carried out at a restriction site present in one of the two antibiotic resistance
genes. E.g, a foreign DNA can be joined at the bam HI site of tetracycline resistance gene in
the vector pBR 322.
E.G E.Coli cloning vector pBR322 diagram

 Letter P – stands for plasmid. B & R – stands for the initials of the two scientist who
developed it. Ori –origin of replication which permits production of multiple copies per cell.
It has two selectable markers (Antibiotic Resistance Genes), that is - Tetracycline (TetR) &
Ampicillin (AmpR). It also contain unique recognition sites for 12 Restriction Enzymes
(endonucleases). Two unique sites: Pst I & Pvu I are present within AmpR gene and Bam
HI, Sal I are within TetR gene. (Refer figure). Rop codes for the proteins involved in the
replication of the plasmid.
 The presence of restriction sites within the markers TetR and AmpR help us to select the
cells transformed with the recombinant pBR 322.
 When enzyme PST I or PVU I is used for the insertion of DNA fragment, the latter is inserted
within gene AmpR. It makes its AmpR non-functional.
  The bacteria cells containing such a recombinant pBR 322 will not be able to grow in the
presence of ampicillin, but will be able to grow in presence of tetracycline.
 In the same way, when restriction enzyme Bam HI or Sal I is used, the DNA is placed within
the gene TetR making the latter non-functional.
 The bacterial cells containing such a recombinant pBR 322 will, therefore be able to grow in
presence of ampicillin, but not in presence of tetracycline.
Selection of Recombinants
The recombinant vectors can be selected by two methods:
1. Antibiotic resistance
2. Colour reaction
ANTIBIOTIC RESISTANCE: The recombinant plasmids lose tetracycline resistance due to
insertion of foreign DNA. It can be selected out from non-recombinants on ampicillin containing
medium. The transformants growing on ampicillin containing medium are then transformed on a
medium containing tetracycline.
The recombinants will grow in ampicillin containing medium but not on that containing tetracycline.
However, non-recombinants will be able to grow on the medium containing both the antibiotics. In
the present case, one antibiotic resistance gene helps in selecting the transformants but the other
antibiotic resistance gene becomes inactivated due to insertion of foreign DNA and helps in selection
of recombinants.
COLOUR REACTION: The selection by antibiotic resistance is a tedious procedure.
An alternative selectable marker developed to differentiate into recombinants and non- recombinants
on the basis of their ability to produce the colour in the presence of a chromogenic substrate. In this
process, a recombinant DNA is inserted in the coding sequence of an enzyme, α- galactosidase. This
causes inactivation of the enzyme; this is called insertional inactivation.
If the plasmid in the bacterium does not have an insert, the presence of a chromogenic substrate
gives blue coloured colonies. The presence of insert result into insertional inactivation of the β-
galactosidase and the colonies do not produce any colour .Such colonies are marked as recombinant
colonies.
VECTORS FOR CLONING GENES IN PLANTS AND ANIMALS
 There are procedures of transferring genes into plants and animals from bacteria and viruses.
Example: Agrobacterium tumifaciens - A pathogen that produces crown galls or plant
tumours in almost all dicot plants.
 The bacterium contains a large Ti-plasmid (tumour inducing plasmid) and is able to transfer
a piece of DNA known as T-DNA to convert normal cells into tumour and direct these
tumour cells to produce the chemicals required by the pathogen. Similarly retroviruses in
 animals including humans are able to change normal cells into cancerous cells. So it enabled
scientists to develop techniques to transfer genes by pathogens in their eukaryotic host.
 The tumour inducing (Ti) plasmid of agrobacterium tumefaciens has now been modified into
a cloning vector which is no more pathogenic to the plants but is still able to use the
mechanism to deliver genes of our interest into variety of plants. Retroviruses have also been
non –pathogenic and are now used to deliver desirable genes into animal cells. Thus, once a
gene or DNA fragment is joined to a suitable vectors, it is transferred into a bacterial ,plant
or animal host, where it undergoes into multiplication.
Agrobacterium tumefaciens (crown tumour)

Competent Host - Host for transformation with Recombinant DNA

 The cell which is capable of taking up alien DNA is called competent host. It is essential
for transformation with recombinant DNA. Since DNA is a hydrophillic molecule, it
cannot pass through membranes so the bacterial cells must be made competent to take up
DNA.
 It is achieved by treating the bacteria with a specific concentration of a divalent cation,
such as calcium, which increases the efficiency with which DNA enters the bacterium
through pores in its cell wall.
 Recombinant DNA can then be forced into such cells by incubating the cells with
recombinant DNA on ice, followed by placing them briefly at 42° C (heat shock) and then
putting them back on ice, this make the bacteria to take up recombinant DNA.
Other methods of introducing DNA into Host Cells
Following are some other methods to introduce alien DNA into host cells:
1. Micro-injection
2. Biolistics or gene gun
3. Disarmed pathogen vectors
Micro-injection
• In this method recombinant DNA is directly injected into the nucleus of animal cell using
micro-needles or micro –pipettes.

Biolistics or Gene Gun

 This method was first developed by Stanford et al at Cornell University, U.S.A. in 1987.
 In this method micro particles (1-2 μm in size) of gold or tungsten coated with DNA are
bombarded with high velocity into the plant cells, using a helium pressure particles gun
device. This technique was developed for plant yet it is also used to insert genes into
animal cell.

Disarmed Pathogen Vectors

 This method uses disarmed pathogen (transformed pathogen incapable of causing diseases)
vectors. These pathogens with recombinant DNA vector, when allowed to infect the cell,
transfer the recombinant DNA into the host.
Processes of Recombinant DNA Technology
The sequence wise steps in recombinant DNA (r DNA) technology includes:
1. Isolation of DNA
2. Fragmentation of DNA by restriction endonucleases
3. Isolation of a desired DNA fragment
 4. Ligation of the DNA fragment into a vector
5. Transferring the recombinant DNA into the host
6. Culturing the host cells in a medium at large scale
7. Extraction of desired products.
Isolation of the Genetic Material (DNA)
 The DNA must be in pure form and free from other macro- molecules when it is to be
cut with restriction enzyme.
 Since the DNA is enclosed within membranes and cell wall, it has to break the cell
open to release DNA and other macromolecules like RNA, protein, polysaccharides and
lipids.
 Therefore, the bacterial cells/plants or animal tissues are treated with enzymes such as
lysozyme (bacteria), cellulose (plant cells), chitinase (fungus) to release the DNA along
with other macromolecules.
 DNA molecules are intertwined with histone proteins, which can be removed by
treating it with enzyme protease. Associated RNA if any, can be removed by treating it
with enzyme ribonuclease.
 The purified DNA, finally precipitates out after the addition of chilled ethanol. The
precipitated DNA can be separated out as fine threads by spooling i.e. winding of fine
threads of DNA on a reel.
 DNA that Separates out can be Removed by Spooling

Spooling =Reel for winding yarn

Cutting of DNA at Specific Locations

 The purified DNA molecules are cut with restriction enzyme by incubating at optimal
conditions which are specific to that enzyme.
 Agarose gel electrophoresis is used to check the progression of restriction enzyme
digestion. Since DNA is a negatively charged molecule, it moves towards anode
(positive electrode). The vector DNA is also cut with same restriction enzyme so as to
get complementary sticky ends.
  After cutting the source DNA and the vector DNA with a specific restriction enzyme,
the cut out “gene of interest” from the source DNA and cut vector having space are
mixed and the enzyme ligase is added.
 The enzyme ligase joins the gene of interest with the cut vector to produce a
recombinant DNA.

Amplification of Gene of Interest using PCR

 The process of making many copies of a gene is called amplification of gene. It is
achieved through a technique called PCR polymerase chain reaction invented by Kary
Mullis (1985).
 It is just like a photocopying a document, in this multiple copies of the gene (DNA) of
interest is synthesized in vitro, using two sets of primers (small chemically synthesised
oligonucleotides that are complementary to the regions of DNA) and the enzyme DNA
polymerase. This technique generates up to billion copies of the desired DNA (or RNA)
segment within few hours.
 PCR involves three steps:
i. Denaturation : The target DNA is heated to a high temperature (usually 94 °c). It
results in separation of the two strands, each single strand of the target DNA acts
as a template for DNA synthesis.
ii. Annealing : In this step, two small chemically synthesised oligonucleotides (that
are complementary to the regions of DNA) primers anneal (or hybridise )to each of
the single stranded template for DNA.
iii. Primer extension (polymerization): In this step a thermostable enzyme Taq DNA
polymerase (isolated form a bacterium, thermus aquaticus) is used to extend
primers using the nucleotides and the genomic DNA as template. Synthesis of new
DNA strand begins in between the primers, using deoxyribonucleotide
triphosphates and Mg2+. The optimum temperature for this polymerization is kept
at 72 °C. The Taq DNA polymerase remains active during the high temperature
induced denaturation of double stranded DNA.

 If the process of replication of DNA is repeated many times, the segment of DNA can
be amplified to approximately billion times (i.e .1 billion copies are made.
 The next PCR amplifications cycle begins as soon as all the stages of previous cycle
end. During PCR operation, the extension product of one cycle serves as a template for
subsequent cycles and each time the amount of DNA doubles.
 Thus, a single template molecule DNA generates 2n molecules at the end of n cycles.
The amplified fragment if desired can now be used to ligate with a vector for further
cloning.
Insertion of Recombinant DNA into the Host cell/organism
 The ligated DNA is introduced into the recipient cells by making them competent to
receive DNA.
 If a recombinant DNA bearing gene for resistance to an antibiotic ampicillin is
transferred into E .Coli cells, the host cells become transformed into ampicillin
resistant cells. If the transformed cells are spread on agar plates containing ampicillin,
only transformants will grow, untransformed recipient cells will die.
 Therefore, due to ampicillin resistance gene, one is able to select a transformed cell in
the presence of ampicillin. In this case, the ampicillin resistance gene is called
“selectable marker”.
Obtaining the Foreign Gene Product
 Most of the recombinant technologies are aimed to produce proteins on large scale.
 The cells containing cloned gene of interest may be grown on a small scale in the
laboratory. The cultures may be used for extracting and purifying the desired protein.
 The cells can also be multiplied in a continuous system (continuous process), where the
used medium is taken out from one side and fresh medium is added from the other side
to maintain the cell in their physiologically most active log or exponential phase i.e,
phase of rapid multiplication of cells .
 Through this type of culturing method, large quantity of biomass is produced which
gives rise to higher yields of desired proteins.
 Small volume cultures cannot yield the product in large quantities, therefore large steel
vessels called bioreactors are employed to obtain the product in large quantities. In a
bio reactor large volumes (100 – 1000 L) of culture can be processed. These are
vessels in which raw materials are biologically converted into specific products,
 individual enzyme etc. using microbial, plant, animal or human cells. A bio reactor
provides the optimal conditions for achieving the desired product by providing optimal
growth conditions such as substrate, salts, vitamins, temperature, pH, oxygen, etc.
 The bioreactors are of different types, however, the stirring type is the most commonly
used type reactor.

BIO REACTOR

 A stirring type bio reactor is usually cylindrical or has a curved base so as to facilitate the
mixing of the reactor contents. It has a stirrer which facilitates even mixing and oxygen
availability throughout the bio reactor.
 The bio reactor has a number of systems such as agitator system, an oxygen delivery
system, a form control system, a temperature control system and pH control system, etc. It
also has a sampling ports so that small volumes of culture can be taken out periodically.
DOWNSTREAM PROCESSING
 The process of separation and purification of a biosynthetic product is called downstream
processing. The products are subjected to series of processes before it is ready for
marketing as a finished product. These processes include separation & purification of the
product. The product has to be formulated with suitable preservatives. In case of drug, such
formulations have to undergo through clinical trial.
 A proper quality control testing is also required for each product.
 Different types of downstream processing and quality control testing are employed for
different types of products.