Biotechnology: Principles and Processes Class 12 Notes

Biotechnology is defined as the broad area of biology which uses both the technology and the application of
living organisms and their components to develop, modify and produce useful products for human welfare.

Principles of Biotechnology
According to modern Biotechnology, the main principles of Biotechnology are:

● Genetic engineering, which is used to modify the DNA of the target organism, thereby changing the
phenotype of the organism.

● Bioprocess engineering, which is the maintenance of sterile conditions to support the growth of large
quantities of desired microbes and other eukaryotic cells which are used for the production of new or
modified biotechnological products such as antibiotics, enzymes, vaccines, etc.
The techniques of genetic engineering mainly include:

1. DNA fragment is isolated from the donor organism.

2. It is inserted into the vector DNA.
3. It is transferred into an appropriate host.
4. Cloning of the recombinant DNA in the host organism.

What is Recombinant DNA technology?

Recombinant DNA technology is also known as Genetic Engineering. It is the process of joining together
two DNA molecules from two different organisms. This is known as the recombinant DNA.
The steps involved in the processes of Recombinant DNA technology are:

1. Isolation of DNA
2. DNA fragmentation using restriction endonucleases
3. Ligation of the desired DNA fragment into the vector
4. Transfer of the recombinant DNA into the host
5. Culture of the transformed cells in a nutrient medium.
6. Extraction of the desired product.

DNA Cloning
DNA cloning is the process of making multiple, identical copies of a piece of DNA. This process requires
cloning vectors which possess the following properties:

● It should be smaller in size but should be able to carry a large DNA insert.

● The cloning vector should have the origin of replication so that it can autonomously replicate in the
host organism.
● It should have a restriction site.

● It should have a selectable marker to screen recombinant organism.

● It should possess multiple cloning sites.

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
plasmid.
(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 to 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 (ii) Polymerase enzymes
(iii) Ligases (iv) Vectors
(v) Competent host organism.

Nuclease- Enzymes which cut DNA and RNA

Exonuclease Endonuclease

X Removes nucleotides from the end.

Y It makes internal cuts. Met------------

Restriction enzymes or ‘molecular scissors’ are used for cutting DNA.

(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.
(iv) 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 letter R is derived from the name of strain.

( c) 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

(v) 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.
(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 read the same on the two strands in 5′ -> 3′
direction as well as 3′ -> 5′ direction. NAYAN
5′ — GAATTC — 3′
3′ — CTTAAG — 5′
(vi) 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.
7. Separation and Isolation of DNA Fragments
(i) The cutting of DNA by restriction’endonucleases results in the fragments of DNA.
(ii) The technique, which separates DNA fragments based on their size is called gel
electrophoresis.
(iii) DNA fragments are negatively charged molecules. They can be separated by
forcing them to move towards the anode under an electric field through a
medium/matrix.
(iv) The most common medium used is agarose, a natural polymer extracted from
sea weeds.
(v) The DNA fragments separate (resolve) according to their size through sieving
effect provided by the agarose gel. The smaller the fragment size, the farther it
moves.

(vi) The separated DNA fragments can be visualised only after staining the DNA
with a compound known as ethidium bromide – ETBr followed by exposure to UV
radiation.
(vii) The DNA fragments can be seen as bright orange coloured bands. These
separated bands are cut out from the agarose gel and extracted from the gel piece.
This is called elution.

(viii) The purified DNA fragments can be used in constructing recombinant DNA by
joining them with cloning vectors.

8. Cloning vectors are the DNA molecules that can 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 X
phage.
(ii) Copy number can be defined as the number of copies of vectors present in a cell.
(iii) Bacteriophages have high number per cell, so their copy number is also high in
genome.
(iv) Plasmids have only one or two copies per cell.
(v) Copy number can vary from 1-100 or more than 100 copies per cell.
(vi) If an alien piece of DNA is linked with bacteriophage or plasmid DNA, its number
can be multiplied equal to the copy number of the plasmid or bacteriophage.

IMP(vii) 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.
(ii) 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 using vector.
• The genes encoding resistance to antibiotics such as ampicillin, chloramphenicol,
tetracycline or kanamycin, etc, are some useful selectable markers for E. coli.

pBR 322 - p= Plasmid ,BR= Boliver Rodriguez , 322 represent the no.of plamid
synthesize by them.

rop= Proteins of replication of plasmid

• 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 the Bam 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 recombinants will grow in ampicillin containing medium but not on that containing tetracycline.
The non-recombinants will grow on the medium containing both the antibiotics.
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, so alternative
selectable markers are developed which differentiate recombinants from non-recombinants on the basis of
their ability to produce colour in the presence of a chromogenic substrate.
-» In this method, a recombinant DNA is inserted within the coding sequence of an enzyme J3-
galactosidase.
-» This results into inactivation of the enzyme, a-galactosidase (insertional inactivation).
-> The bacterial colonies whose plasmids do not have an insert, produce blue colour, but others do not
produce any colour, when grown on a chromogenic substrate.
(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.
(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.
-» Agrobacterium tumefaciens is a pathogen of several dicot plants.
-» It delivers a piece of DNA known as T-DNA in the Ti plasmid which can transforms normal plant cells
into tumour cells to produce chemicals
required by pathogens.
• Retrovirus, adenovirus, papillomavirus are also now used as cloning vectors in animals because of their
ability to transform normal cells into cancerous cells.
9. 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,
(i) Competency is the ability of a cell to take up foreign DNA.
(ii) 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 (heat
shock) and then putting it back on ice. This is called heat shock treatment.
• 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 recombinant
DNA into the host.eg. Agrobacterium tumefaciens, Retrovirus, adenovirus, papillomavirus

Biolistic method

PROCESSES OF RECOMBINANT DNA TECHNOLOGY

Recombinant DNA technology involves several steps in specific

sequence such as isolation of DNA, fragmentation of DNA by

restriction endonucleases, isolation of a desired DNA fragment,

ligation of the DNA fragment into a vector, transferring the

recombinant DNA into the host, culturing the host cells in a

medium at large scale and extraction of the desired product.

Let us examine each of these steps in some details.

11.3.1 Isolation of the Genetic Material (DNA)

Recall that nucleic acid is the genetic material of all organisms

without exception. In majority of organisms this is

deoxyribonucleic acid or DNA. In order to cut the DNA with

restriction enzymes, it needs to be in pure form, free from other

macro-molecules. Since the DNA is enclosed within the

membranes, we have to break the cell open to release DNA along

with other macromolecules such as RNA, proteins,

polysaccharides and also lipids. This can be achieved by treating

the bacterial cells/plant or animal tissue with enzymes such as

lysozyme (bacteria), cellulase (plant cells), chitinase (fungus).

You know that genes are located on long molecules of DNA

interwined with proteins such as histones. The RNA can be removed by

treatment with ribonuclease whereas proteins can be removed by

treatment with protease. Other molecules can be removed by appropriate

treatments and purified DNA ultimately precipitates out after the addition

of chilled ethanol. This can be seen as collection of fine threads in the

suspension (Figure 11.5).

Cutting of DNA at Specific Locations

Restriction enzyme digestions are performed by incubating purified DNA

molecules with the restriction enzyme, at the optimal conditions for that

specific enzyme. Agarose gel electrophoresis is employed to check the

progression of a restriction enzyme digestion. DNA is a negatively charged

molecule, hence it moves towards the positive electrode (anode)

(Figure 11.3). The process is repeated with the vector DNA also.

The joining of DNA involves several processes. After having cut the

source DNA as well as the vector DNA with a specific restriction enzyme,

the cut out ‘gene of interest’ from the source DNA and the cut vector with

space are mixed and ligase is added. This results in the preparation of
recombinant DNA.

11.3.3 Amplification of Gene of Interest using PCR

PCR stands for Polymerase Chain Reaction. In this reaction, multiple

copies of the gene (or DNA) of interest is synthesised in vitro.

How does PCR work?

PCR mimics what happens in cells when DNA is copied (replicated) prior to cell division, but it
is carried out in controlled conditions in a laboratory. The machine that is used is simply called
a PCR machine or a thermocycler. Test tubes containing the DNA mixture of interest are put
into the machine, and the machine changes the temperature to suit each step of the process.

Standard ingredients in the mixture are:

● the DNA segment of interest

● specific primers
● heat-resistant DNA polymerase enzyme
● the four different types of DNA nucleotides
● the salts needed to create a suitable environment for the enzyme to act.

Step 1: Denaturation at 94-96 0

C

As in DNA replication, the two strands in the DNA double helix need to be separated.

The separation happens by raising the temperature of the mixture, causing the hydrogen bonds between the
complementary DNA strands to break. This process is called denaturation.

Step 2: Annealing at 40 -60 0C

Primers bind to the target DNA sequences and initiate polymerisation. This can only occur once the
temperature of the solution has been lowered. One primer binds to each strand.

Step 3: Extension at 72 0C

New strands of DNA are made using the original strands as templates. A DNA polymerase enzyme joins free
DNA nucleotides together. This enzyme is often Taq polymerase, an enzyme originally isolated from a
thermophilic bacteria called Thermus aquaticus. The order in which the free nucleotides are added is
determined by the sequence of nucleotides in the original (template) DNA strand.

The result of one cycle of PCR is two double-stranded sequences of target DNA, each containing one newly
made strand and one original strand. If the process of replication

of DNA is repeated many times, the segment of DNA can be amplified

to approximately billion times

Figure 11.6 Polymerase chain reaction (PCR) : Each cycle has three steps:

(i) Denaturation;

(ii) Annealing-sets of primers (small chemically synthesised oligonucleotides that are

complementary to the regions of DNA) and the enzyme DNA polymerase.

(iii) Extension-The enzyme extends the primers using the nucleotides provided in the

reaction and the genomic DNA as template. 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. Such

repeated amplification is achieved by the use of a thermostable DNA

polymerase (isolated from a bacterium, Thermus aquaticus), which

remain active during the high temperature induced denaturation of

double stranded DNA. The amplified fragment if desired can now be

used to ligate with a vector for further cloning (Figure11.6).

11.3.4 Insertion of Recombinant DNA into the Host

Cell/Organism

There are several methods of introducing the ligated DNA into recipient

cells. Recipient cells after making them ‘competent’ to receive, take up

DNA present in its surrounding. So, if a recombinant DNA bearing gene

for resistance to an antibiotic (e.g., ampicillin) is transferred into E. coli

cells, the host cells become transformed into ampicillin-resistant cells. If

we spread the transformed cells on agar plates containing ampicillin, only

transformants will grow, untransformed recipient cells will die. Since, due

to ampicillin resistance gene, one is able to select a transformed cell in the

presence of ampicillin. The ampicillin resistance gene in this case is called

a selectable marker.

11.3.5 Obtaining the Foreign Gene Product

When you insert a piece of alien DNA into a cloning vector and transfer it

into a bacterial, plant or animal cell, the alien DNA gets multiplied. In

almost all recominant technologies, the ultimate aim is to produce a

desirable protein. Hence, there is a need for the recombinant DNA to be

expressed. The foreign gene gets expressed under appropriate conditions.

The expression of foreign genes in host cells involve understanding many

technical details.

After having cloned the gene of interest and having optimised the

conditions to induce the expression of the target protein, one has to

consider producing it on a large scale. Can you think of any reason

why there is a need for large-scale production? If any protein encoding

gene is expressed in a heterologous host, is called a recombinant

protein. The cells harbouring cloned genes of interest may be grown

on a small scale in the laboratory. The cultures may be used for

extracting the desired protein and then purifying it by using different

separation techniques.

The cells can also be multiplied in a continuous culture system wherein

the used medium is drained out from one side while fresh medium is

added from the other to maintain the cells in their physiologically most

active log/exponential phase. This type of culturing method produces a

larger biomass leading to higher yields of desired protein.

Small volume cultures cannot yield appreciable quantities of products.

To produce in large quantities, the development of bioreactors, where

large volumes (100-1000 litres) of culture can be processed, was required.

Thus, bioreactors can be thought of as vessels in which raw materials are

biologically converted into specific products, individual enzymes, etc.,

using microbial plant, animal or human cells. A bioreactor provides the

optimal conditions for achieving the desired product by providing

optimum growth conditions (temperature, pH, substrate, salts, vitamins,

oxygen).

The most commonly used bioreacters are of stirring type, which are

shown in Figure 11.7.

Figure 11.7 (a) Simple stirred-tank bioreactor; (b) Sparged stirred-tank bioreactor through which
sterile air bubbles are sparged

(A stirred-tank reactor is usually cylindrical or with a curved base to

facilitate the mixing of the reactor contents. The stirrer facilitates even

mixing and oxygen availability throughout the bioreactor. Alternatively

air can be bubbled through the reactor. If you look at the figure closely

you will see that the bioreactor has an agitator system, an oxygen delivery

system and a foam control system, a temperature control system, pH

control system and sampling ports so that small volumes of the culture

can be withdrawn periodically.

11.3.6 Downstream Processing

After completion of the biosynthetic stage, the product has to be subjected

through a series of processes before it is ready for marketing as a finished product. The processes include
separation and purification, which are

collectively referred to as downstream processing. The product has to be

formulated with suitable preservatives. Such formulation has to undergo

thorough clinical trials as in case of drugs. Strict quality control testing

for each product is also required. The downstream processing and quality

control testing vary from product to product.