Biotechnology: Principles and Processes

1. Biotechnology
➢ Biotechnology deals with microorganisms, plant or animal cells or their enzymes to produce
products and processes useful to humans.
➢ The term “Biotechnology” was given by Karl Ereky (1919).
➢ According to European Federation of Biotechnology (EFB), biotechnology is the integration of
natural science and organisms, cells, parts thereof, and molecular analogues for products
and services.

2. Principles of Biotechnology
➢ The two core techniques that developed modern biotechnology are:
(i) Genetic engineering which is modification of chemical nature of DNA/RNA and their
introduction into another host organism, to change the phenotypic characters of the
host.
(ii) Sterilisation methods to maintain growth and manipulation of only the desired
microbes or cells in large quantities, for the manufacture of biotechnological
products like antibiotics, vaccines, enzymes, etc.
➢ The basic steps in genetic engineering include:
(i) identification of DNA with desirable genes.
(ii) introduction of the DNA into host to form recombinant DNA (rDNA).
(iii) maintenance of DNA in host and gene cloning.
(iv) gene transfer.
➢ In 1972, Stanley Cohen and Herbert Boyer constructed the first recombinant DNA.
➢ Herbert Boyer worked on restriction enzymes of E. coli which cut DNA in particular fashion
and produce sticky ends on both the strands. These restricted ends were ligated with desired
pieces of DNA.
➢ Stanley Cohen studied plasmid DNA floating freely in cytoplasm of bacterial cells. He also
developed a method of removing plasmids from the cell and reinserting them in other cells.
➢ They isolated antibiotic resistant gene from plasmid of bacteria and then linked the gene
with plasmid and incorporated into E coli, where it could replicate using the new host’s DNA
polymerase enzyme and make multiple copies.
➢ Steps carried out in constructing first recombinant DNA:
(i) A gene encoding antibiotic resistance in the native plasmid of Salmonella
typhimurium V. was identified. Plasmid is an autonomously replicating circular extra-
chromosomal DNA.
(ii) The desired DNA was cut at specific locations by restriction enzymes.
(iii) The cut DNA was linked to plasmid DNA and transferred to E. coli for gene
multiplication.

3. Tools of Recombinant DNA Technology

➢ The key tools required for the recombinant DNA technology are:
(i) Restriction enzymes (ii) Polymerase enzymes (iii) Ligases (i) Vectors (v) Host organism/cell.
4. Restriction Enzymes
➢ The restriction enzymes are called “molecular scissors” and are responsible for cutting DNA.
➢ They are present in bacteria to provide a type of defence mechanism called the “restriction–
modification system”.
➢ The first restriction endonuclease, HindII, was isolated by Smith, Wilcox and Kelley (1968)
from Haemophilus influenzae bacterium. It was used to cut DNA molecules at a particular
point by recognising a specific sequence of six base pairs, known as the recognition
sequence. Naming of Restriction Enzymes
➢ The first letter is derived from the genus name and the next two letters from the species
name of the prokaryotic cell from where the enzymes are extracted. The roman numbers,
following the name, show the order in which the enzymes were isolated from the bacterial
strain.
➢ For example, EcoRI is derived from Escherichia coli RY13, HindII from Haemophilus influenzae
Rd, BamHI from Bacillus amyloliquefaciens H, EcoRII from E. coli R245, etc.
➢ Restriction enzymes belong to a class of enzymes called nucleases and are of two types:
(i) Exonucleases—cut DNA at the ends.
(ii) Endonucleases—cut at specific positions within the DNA.
➢ The recognition sequences of endonucleases are palindromic, i.e., the sequence of base
pairs read the same on both the DNA strands, when orientation of reading is kept same, e.g.,
5′------- G A A T T C-------- 3′
3′------- C T T A A G-------- 5′

Mechanism of Action of Endonucleases

➢ Every endonuclease inspects the entire DNA sequence for the palindromic recognition
sequence.
➢ On finding the palindrome, the endonuclease binds to the DNA.
➢ It cuts the opposite strands of DNA in the sugar–phosphate backbone; a little away from the
centre of the palindrome sites but between the same bases on both strands.
➢ This results in the formation of single stranded overhanging stretches at the end of each
strand called sticky ends.
➢ The sticky ends facilitate the action of the enzyme DNA ligase by readily forming hydrogen
bonds with complementary strands.
➢ In genetic engineering, DNA from different sources is cut with the same restriction enzymes
so that both DNA fragments have same kind of sticky ends.
➢ These sticky ends are complementary to each other and thus can be joined by DNA ligase
(end-to-end)
5. Separation and Isolation of DNA Fragments (Gel Electrophoresis)
➢ Gel electrophoresis is a technique for separating DNA fragments based on their size.
➢ Firstly, the sample DNA is cut into fragments by restriction endonucleases.
➢ The DNA fragments being negatively charged can be separated by forcing them to move
towards the anode under an electric field through a medium/matrix.
➢ Commonly used matrix is agarose, which is a natural linear polymer of D-galactose and 3, 6-
anhydro-L-galactose which is extracted from sea weeds.

➢ The DNA fragments separate-out (resolve) according to their size because of the sieving
property of agarose gel. Hence, smaller the fragment size, the farther it will move.
➢ The separated DNA fragments are visualised after staining the DNA with ethidium bromide
followed by exposure to UV radiation.
➢ The DNA fragments are seen as orange-coloured bands.
➢ The separated bands of DNA are cut out and extracted from the gel piece. This step is called
elution.
➢ The purified DNA fragments are used to form recombinant DNA which can be joined with
cloning vectors.

6. Cloning Vectors
➢ The vectors are the DNA molecules that can carry a foreign DNA segment into the host cell.
➢ Vectors may be: (a) Plasmids: These are autonomously replicating circular extra-
chromosomal DNA. (b) Bacteriophages: These are viruses that infect bacteria.
Bacteriophages because of their high number per cell have very high copy number within
bacterial cells.
➢ Copy number: It is defined as the number of copies of vectors present in a cell. It varies from
1–100 copies per cell.
➢ The best-known vector is the plasmid vector.
➢ pBR322 is the first artificial cloning vector developed in 1977 by Boliver and Rodriguez from
E. coli plasmid.

➢ The following features are required to facilitate cloning into a vector:

(i) Origin of replication (ori)
• This is a DNA sequence that is responsible for initiating replication. Any piece
of DNA when linked to this sequence can replicate within the host cells.
• ori also controls the copy numbers of the linked DNA. For many copies of
target DNA, it should be cloned in a vector whose origin supports high copy
number.
(ii) Selectable marker
• It helps to select the host cells which contain the vector (transformants) and
eliminate the non-transformants.
• Transformation is defined as the procedure by which a piece of DNA is
introduced into a bacterial cell.
• Genes encoding resistance to antibiotics like ampicillin, chloramphenicol,
tetracycline or kanamycin, are useful selectable markers for E. coli.
• The normal E. coli cells do not carry resistance against any of these
antibiotics.
(iii) Cloning sites
• To link the alien DNA, the vectors require very few (mostly single)
recognition sites for the restriction enzymes.
• More than one recognition sites within the vector, can complicate the gene
cloning as it will generate several fragments.
• Ligation of alien DNA can be carried out at a restriction site present in one of
the two antibiotic resistance genes.
(iv) Vectors for cloning genes in plants and animals
• There are several vectors which are used for cloning genes in plants and
animals.
• In plants, the tumour inducing plasmid (Ti) of Agrobacterium tumefaciens is
used as a cloning vector.
• A. 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 against
pathogens.
 • Retrovirus, adenovirus, papillomavirus is also now used as cloning vectors in
animals because of their ability to transform normal cells into cancerous
cells. They are first disarmed and then used to transfer desirable genes to
animal cells.
➢ Selection of recombinants formed can be done by one of the following methods:
(a) Inactivation of antibiotics
• If a foreign DNA ligates at the BamHI site of tetracycline resistance gene
in the vector pBR322, the recombinant plasmid loses the tetracycline
resistance due to insertion of foreign DNA.
• It can still 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 to a medium containing tetracycline.
• The recombinants can grow in ampicillin containing medium but not on
that containing tetracycline whereas non-recombinants can grow on the
medium containing both the antibiotics and thus recombinants are
selected.
(b) Alternative Selectable Marker (Insertional inactivation)
• On the basis of colour production in the presence of chromogenic
substrate, the recombinants and non-recombinants can also be
differentiated.
• Here, a recombinant DNA is inserted within the coding sequence of an
enzyme β-galactosidase, which results into inactivation of the enzyme
and hence there is no conversion of substrate to products.
• Hence, the bacterial colonies having transformed plasmid, shows no
colouration while those without inserted plasmid form blue colour
colonies.

7. Competent Host (For Transformation with Recombinant DNA)

➢ DNA being a hydrophilic molecule, cannot pass through cell membranes.
➢ Therefore, the bacteria should be made competent to accept the DNA molecules.
➢ Competency is the ability of a cell to take up foreign DNA.
➢ The cell is made competent by the following methods: (i) Chemical method (ii) Physical
method
(i) Chemical method
• The cell is treated with specific concentration of a divalent cation such as
calcium to increase pore size in cell wall.
• The cells are 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.
• The bacteria now take up the recombinant DNA.
(ii) Physical methods
The physical methods include
• Micro-injection method: Recombinant DNA is directly injected into the
nucleus of an animal cell of micro-pipettes.
• Biolistic or gene gun method: Cells are bombarded with high velocity micro-
particles of gold or tungsten coated with DNA in plants.
 ➢ Disarmed pathogen vectors are also used to transfer rDNA like Agrobacterium tumefaciens.

8. Process of Recombinant DNA Technology

➢ Recombinant DNA technology involves the following steps:
(i) Isolation of DNA
(ii) Fragmentation of DNA by restriction endonucleases
(iii) Isolation of a desired DNA fragment
(iv) Amplification of the gene of interest
(v) Ligation of the DNA fragment into a vector
(vi) Insertion of recombinant DNA into the host
(vii) Culturing the host cells on a suitable medium at a large scale
(viii) Extraction of the desired gene product
(ix) Downstream processing of the products as finished product, ready for marketing

(i) Isolation of the genetic material (DNA)

• To cut DNA with restriction enzymes DNA should be purified, free from other
macromolecules.
• The bacterial/plant/animal cell is broken down by enzymes to release DNA, along
with RNA, proteins, polysaccharides and lipids.
• Bacterial cell is treated with enzyme lysozyme.
• Plant cell is treated with enzyme cellulase.
• Fungal cell is treated with chitinase
• RNA is removed by treatment with ribonuclease and proteins are removed by
treatment with protease.
• After several treatments, the purified DNA is precipitated by adding chilled ethanol.
(ii) Cutting of DNA at specific locations
• The DNA is cut using restriction enzymes.
• The purified DNA is incubated, with the specific restriction enzyme at conditions
optimum for the enzyme to act.
 • Process is repeated with vector DNA also.
(iii) Isolation of desired DNA fragment
• Using agarose gel electrophoresis, the activity of the restriction enzymes can be
checked.
• Since the DNA is negatively charged, it moves towards the positive electrode or
anode and in the process, DNA fragments separate out based on their sizes.
• The desired DNA fragment is eluted out.
(iv) Amplification of gene of interest using PCR
• The Polymerase Chain Reaction (PCR) is a reaction in which amplification of specific
DNA sequences is carried out in vitro.
• This technique was developed by Kary Mullis in 1985, and for this, he received Nobel
Prize for Chemistry in 1993.
• Requirements for PCR:
(a) DNA template: The double-stranded DNA that needs to be amplified.

(b) Primers: Small chemically synthesised oligonucleotides of about 10–18

nucleotides that are complementary to a region of template DNA.
(c) Enzyme: Two commonly used enzymes are Taq polymerase (isolated from
thermophilic bacterium, Thermus aquaticus) and Vent polymerase (isolated from
Thermococcus litoralis).
• PCR is carried out in the following three steps:
(a) Denaturation — The double-stranded DNA is denatured by subjecting it to high
temperature of 94°C for 15 seconds. Each separated single stranded strand now acts
as template for DNA synthesis.
(b) Annealing — Two sets of primers are added which anneal to the 3′ end of each
separated strand. — Primers act as initiators of replication.
(c) Extension — DNA polymerase extends the primers by adding nucleotides
complementary to the template provided in the reaction. — A thermostable DNA
polymerase (Taq polymerase) is used in the reaction which can tolerate the high
 temperature of the reaction. — All these steps are repeated many times to obtain
several copies of desired DNA.
(v) Ligation of DNA fragment into a vector
• The vector DNA and source DNA are cut with the same endonuclease to obtain sticky
ends.
• These are then ligated by mixing vector DNA, gene of interest and enzyme DNA
ligase to form a recombinant DNA.
(vi) Insertion of recombinant DNA into the host cell/organism
• Introduction of ligated DNA into recipient cells occurs by several methods, before
which the recipient cells are made competent to receive the DNA.
• If recombinant DNA carrying antibiotic resistance (e.g., ampicillin) is transferred into
E. coli cells, the host cell is transformed into ampicillin-resistant cells.
• The ampicillin resistant gene in this case is called a selectable marker.
• On growing transformed cells on agar plates containing ampicillin, only
transformants will grow and others will die.

(vii) Culturing the host cells

• The transformed host cells are grown in appropriate nutrient medium at optimal
conditions.
• The DNA gets multiplied and expresses itself to form desired product.
(viii) Extraction of desired gene product
• When a protein encoding gene is expressed in a heterologous host, it is called a
recombinant protein.
• The cells having genes of interest can be grown on a small scale or on a large scale.
• On small scale, the cells are grown on cultures in laboratory and then the expressed
protein is extracted and purified by different separation techniques.
• On large scale, the cells are grown in a continuous culture system in which fresh
medium is added from one side, to maintain cells in exponential growth phase and
the desired protein is collected from the other side.
• In large scale method, larger biomass is produced which leads to high yield.
(ix) Downstream processing
• All the processes to which a product is subjected to before being marketed as a
finished product are called downstream processing.
• It includes:
(a) Separation of the product from the reactor.
(b) Purification of the product.
(c) Formulation of the product with suitable preservatives.
(d) Quality control testing and clinical trials in case of drugs.

9. Bioreactors
➢ Bioreactors are vessels of large volumes (100–1000 litres) in which raw materials are
biologically converted into specific products.
➢ It provides all the optimal conditions for achieving the desired product by providing optimal
growth conditions like temperature, pH, substrates, salts, vitamins and oxygen.
➢ Stirred-tank bioreactors are commonly used bioreactors.
➢ These are cylindrical with curved base to facilitate
(i) proper mixing of the contents,
(ii) maintain oxygen availability throughout the bioreactor.
➢ Stirred tank reactor has
(i) better temperature and pH control,
(ii) foam control system to prevent foam and shearing damage to cells due to agitation,
(iii) system sterilisation, and
(iv) provision to withdraw small volumes of cultures periodically.

➢ A bioreactor has the following components:

(i) An agitator system
(ii) An oxygen delivery system
(iii) Foam control system
(iv) Temperature control system
(v) pH control system
(vi) Sampling ports to withdraw cultures periodically
➢ The stirrer mixes the contents and makes oxygen available throughout the bioreactor.
➢ Sparged stirred-tank reactor is a stirred type reactor in which air is bubbled.