BIOTECHNOLOGY PRINCIPLES AND PROCESSES

Objectives

 Biotechnology deals with techniques of using live organisms or enzymes from

organisms to produce products and processes useful to man.
 Microbe mediated processes like making of curd, bread ,vine etc are considered as primitive
form of biotechnology.
 Nowadays many processes/techniques are included under biotechnology. For example, in
vitro fertilisation leading to a „test-tube‟ baby, synthesising a gene and using it, developing a
DNA vaccine or correcting a defective gene, are all part of biotechnology.
 The European Federation of Biotechnology (EFB) has defined biotechnology as „The
integration of natural science and organisms, cells, parts thereof, and molecular analogues
for products and services.
PRINCIPLES OF BIOTECHNOLOGY:

1. GENETIC ENGINEERING:
introduce these
into host organisms and thus change the phenotype of the host organism.
2. BIOPROCESS ENGINEERING:
 Maintenance of sterile ambience in chemical engineering processes to enable growth of only
the desired microbe / eukaryotic cell in large quantities for the manufacture of biotechno-
logical products.
 Traditional hybridization procedures used in plant and animal breeding, very often lead to
inclusion and multiplication of undesirable genes along with the desired genes.
 In genetic engineering the genes of desirable qualities are taken from an organism and
transfer it to the host organism. So we will get an organism having desirable qualities.
 The techniques of genetic engineering include creation of recombinant DNA, use of gene
cloning and gene transfer.
 In a chromosome there is a specific DNA sequence called the origin of replication, which is
responsible for initiating replication.
 Therefore, for the multiplication of any alien piece of DNA in an organism, it needs to be a
part of a chromosome which has a specific sequence known as „origin of replication’.
 Thus, an alien DNA is linked with the origin of replication, can replicate and multiply itself
in the host organism.
 This is known as Cloning or making multiple identical copies of any template DNA.
RECOMBINANT DNA TECHNOLOGY
 It is introduced by Stanley Cohen and Herbert Boyer in 1972.
 They isolated an antibiotic resistant gene from Salmonella typhIMURIUM and introduced to
another bacterium called Escherichia coli.
 They cut the antibiotic resistant gene from Salmonella by using restriction endonuclease
enzyme.
 They used plasmids as vectors to transfer this alien DNA to E,coli.
 The linking of antibiotic resistant gene to plasmid is done by using DNA ligase enzyme.
 By using DNA polymerase enzyme it is able to multiply the copies of antibiotic resistance
gene. This ability to multiply copies of a gene is called Gene Cloning.
1 Prepared by Ismail Parambath,KKM Govt.HSS,Orkatteri,Kozhikkode

Downloaded from www.hssreporter.com

  Three Basic steps in rDNA technology:
o 1.Identification of DNA with desirable genes
o 2.Introduction of the identified DNA into the host
o 3.Maintenance of introduced DNA in the host and transfer of the DNA to its progeny.
TOOLS OF RECOMBINANT DNA TECHNOLOGY:
1. Restriction enzymes 2.Polymerase enzymes 3.Ligase enzymes 4.Clonig vectors
5.Competent host (For transformation With Recombinant DNA)
Restriction Enzymes (Molecular Scissors):
 Restriction enzymes are the enzymes produced by certain bacteria and they have the property of
cleaving DNA molecule at specific base sequences.
 Bacterium produces a restriction enzyme to defend against bacterial viruses call bacteriophages,
or phages.
 The restriction enzyme prevents replication of the phage DNA by cutting it into many pieces.
 The restriction enzymes cut DNA at specific base pair sequence, and this specific base sequence
is known as the recognition sequence.
 The first discovered restriction endonuclease - Hind II Nomenclature of Restriction Enzymes
o The naming of restriction enzymes are based on their origin and type of action
For e.g. Eco RI
o E - The first letter of the enzyme name comes from the first letter of the genus of the
prokaryotic cell from which enzyme were isolated (here „E‟ stands for Escherichia)
o Co - The second two letters come from the species name. („co‟ stands for coli)
o R- Third letter indicates first letter of name of strain(„R‟ stands for RY 13 strain of bacteria
from which the enzyme obtained)
o I (roman numeral one)- Indicates the order of discovery of enzyme
 Restriction enzymes belong to a larger class of enzymes called Nucleases.
 These are of two kinds; Exonucleases and Endonucleases.
 Exonucleases remove nucleotides from the ends of the DNA whereas, endonucleases make cuts at
specific position within the DNA.
 Each restriction endonuclease recognizes a specific palindromic nucleotide sequences in the DNA.
 Palindromes are groups of letters that form the same words when read both forward and
backward.eg. “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.
 The recognition site of Eco,RI is given below ; it is a palindromic sequence
- G A A T T C -
- C T T A A G –
 Restriction enzymes cut the strand of DNA a little away from the center of the palindrome sites,
but between the same two bases on the opposite strands.
 This leaves single stranded portions at the ends called Sticky ends.

2 Prepared by Ismail Parambath,KKM Govt.HSS,Orkatteri,Kozhikkode

Downloaded from www.hssreporter.com

 The stickiness of the strands facilitates the action of the enzyme DNA ligase.
 For e.g. EcoRI cut the DNA between G and A only when the sequence GATTC present in the DNA.
It is the recognition site of EcoRI

 Restriction endonucleases are

used in genetic engineering to
form recombinant molecules
 of DNA,which are composed of
DNA from different sources or
genome.

restriction enzyme the
resultant DNA fragments have
the same kind
 of Sticky-ends and can be
joined together using DNA
ligases.

Separation and Isolation of DNA fragments (DNA of interest):

 The cutting of DNA by restriction endonucleases results in the fragments of DNA.
 These fragments can be separated by a technique known as Gel Electrophoresis.
 Negatively charged DNA fragments can be separated by forcing them to move towards the anode
under an electric field through a medium.
 Nowadays most commonly used matrix/medium is Agarose Gel,which is a natural polmer extracted
from sea-weeds.
 The DNA fragments are separated according to their size through sieving effect provided by the
agarose .
 The particles with a smaller size have been reported to move faster and farther away from the
well.
3 Prepared by Ismail Parambath,KKM Govt.HSS,Orkatteri,Kozhikkode

Downloaded from www.hssreporter.com

 As a result the molecules are separated by size.
 Electrophoresis enables you to distinguish DNA
fragments of different lengths.
 The separated DNA fragments can be visualized
only after staining the DNA with Ethidium
bromide followed by exposure to UV radiation.
 Now DNA fragments appear bright orange
coloured bands.
 The separated bands of DNA are cut out from
the agarose gel and extracted from the gel
 piece. This step is known as Elution.

Cloning Vectors (Vehicles for Cloning):
 Vector serves as a vehicle to transfer a foreign gene (DNA sequence) into a given host cell.
 It has the ability to self replicate and integrate into the host cell
Salient features of a Vector:
o origin of replication (ori) so that it is able to multiply within the host
cell.
o It should have restriction sites for the insertion of the target DNA.
o It should incorporate a selectable marker (antibiotic resistance gene), which will allow to
select those host cells that contain the vector from amongst those which do not.
o Some of the commonly used vectors are Plasmids, Bacteriophages, Plant or animal viruses etc
o Plasmids and bacteriophages have the ability to replicate within bacterial cells independent
of the control of chromosomal DNA.
The characteristic features of a plasmid Vectors are

o Any piece of DNA when linked to this

sequence can be made replicate within the
host cells.
o This sequence is responsible for controlling
the copy number of the linked DNA.
2.Selectable Markers
o A vector requires a selectable marker which
helps in identifying and eliminating
transformants from non-transformants.
o Transformation is a procedure through which
a piece of DNA is introduced in a host
Bacterium.
1.Origin of Replication o Normally, the genes encoding resistance to
o It is the sequence from where replication antibiotic such as ampicillin, chloramphenicol,
starts. tetracycline or kanamycin etc. are considered
useful selectable marker for E.coli

4 Prepared by Ismail Parambath,KKM Govt.HSS,Orkatteri,Kozhikkode

Downloaded from www.hssreporter.com

 3.Cloning Sites
o A vector consists of some recognition sites where the alien DNA joins.
o The ligation of alien DNA is carried out at a restriction site present in one of the two
antibiotic resistance genes, for e.g. we can ligate a foreign DNA at the Bam H1 site of
tetracycline resistance gene in the vector pBR 322
o The recombinant plasmids will lose tetracycline resistance due to insertion of foreign DNA
but can still be selected out from non-recombinant ones by plating the transformants on
tetracycline containing medium.
o The transformants growing on ampicillin containing medium are then transferred on a
medium containing tetracycline.
o The recombinants will grow in ampicillin containing medium but not on that containing
tetracycline.
o But, non- recombinants will grow on the medium containing both the antibiotics.
o In this case, 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.
Insertional inactivation:
 Recently, alternative selectable markers have been developed which differentiate
recombinants from non-recombinants on the basis of their ability to produce colour in the
presence of a chromogenic substrate.
 If a bacterial plasmid having a gene for β-galactdosidase enzyme, the bacteria will produce that
enzyme into the medium where it is grown.
 If this medium contain a chromogenic substrate the enzyme will react with this and a blue colour is
produced.
 When a foreign gene is inserted into the β-galactosidase site of the bacterial plasmid, the
bacteria will lose the ability to produce the β-galactosidase enzyme.
 Presence of insert results into insertional inactivation of the β-galactosidase gene and the
colonies do not produce any colour,these are identified as recombinant colonies
 So the colonies of the bacteria having a foreign gene insert can be can be identified by noticing
the colour.This process is called insertional inactivation.
Vectors for cloning genes in plants and animals :
 AgrobacteRIUM tUMefaciens (pathogen of dicot plant) is able to deliver a piece of DNA known as
„T-DNA” to transform normal plant cells into a tumorous one.
 Agrobacterium modify these tumor cells to produce the chemicals required by the pathogen.
 The tumor inducing (Ti) plasmid of Agrobacterium tumefaciens has been modified into cloning
vector having no more pathogenic to plant.
 Retroviruses in animals have the ability to transform normal cells into cancerous cells.
 Nowadays retrovirus have been disarmed and are used to transfer desirable genes into animal
cells.
Competent Host (Introduction of recombinant DNA into host cells):
 In r-DNA technology, the most common method to introduce r-DNA into living cells is
transformation, during which cells take up DNA from the surrounding environment.
5 Prepared by Ismail Parambath,KKM Govt.HSS,Orkatteri,Kozhikkode

Downloaded from www.hssreporter.com

 At first the bacterial cells must made competent to take up the foreign DNA .
 This is done by treating them with a specific concentration of divalent cations like calcium.
 It increases the efficiency of bacteria to take DNA through the pores on the cell wall.
 The recombinant DNA is then forced into bacterial cell by incubating the cells with rDNA
on ice, followed by forcing them briefly at 420C (heat shock) and then putting back on ice.
 This enables the bacteria to take up the recombinant DNA.
Micro injection
o The host cell is made immobilized and then recombinant DNA is injected into the nucleus
of the host cell. (It is done with the help of a micro needle).
o This technique is now used to produce transgenic animals.
Gene Gun
o It is the new technology where vector less direct gene transfer is made in plants.
o High velocity micro particles of gold or tungsten coated with DNA are bombarded on the
host cells. This method is known as biolistics or gene gun.
PROCESSES OF REOMBINANT DNA TECHNOLOGY:
 Recombinant DNA technology involves several steps in specific sequence such as
1)Isolation of the Genetic material
2)Cutting of DNA at specific locations
3)Amplification of Gene of interest using PCR
4)Insertion of Recombinant DNA Into the Host cell /organism
5)Obtaining Foreign gene product
6)Down stream Processing d
1.Isolation of the Genetic material
 DNA or RNA may be the genetic material; in majority of the organisms DNA is the genetic
material
 The cells are broken and opened to release DNA along with other macromolecules such as
RNA, proteins, polysaccharides and lipids.
 It is done by treating the cells with enzymes such as lysozyme (bacteria), cellulase (plant
cells) or chitinase (fungus).
 The RNA can be removed by treatment with ribonuclease whereas proteins can be removed by
treatment with protease.
 After the treatment of appropriate enzymes the DNA can be precipitated by adding chilled
ethanol
 DNA can be seen as collection of fine threads in the suspension.
 Use spooling to extract the precipitated DNA. Spooling involves winding the fine threads
of DNA on to a reel.
Cutting of DNA at specific locations
 For cutting the desired DNA or gene, the purified DNA molecules are incubated with the
restriction endonuclease enzymes.
 The agarose gel electrophoresis is done to analyze the progression of a restriction enzyme
digestion.
 DNA is a negatively charged molecule so it moves towards the positive electrode (anode).
 The vector DNA is also cut by using the same restriction enzyme .
6 Prepared by Ismail Parambath,KKM Govt.HSS,Orkatteri,Kozhikkode

Downloaded from www.hssreporter.com

 After cutting the DNA as well as vector DNA with restriction enzyme, the desired gene
and cut vector are mixed together and DNA ligase enzyme is added to join the two DNA
molecules.
 This results in the formation of recombinant DNA.
3. Amplification of Gene of interest using PCR
 PCR stands for polymerase chain reaction. In this reaction, multiple copies of the gene of
interest can be synthesized in vitro under three steps

a.Denaturation
o In this process the double stranded DNA
is converted into the single stranded DNA.
o It is normally achieved by heating.
b.Annealing.
o The two sets of primers bind to their
complementary sequences on single
stranded DNA.
o Here primers are single-strand sequences
of DNA around 20 to 30 bases in length.
o They serve as the starting point for the
 synthesis of DNA
c.Extension
o The enzyme DNA polymerase extends the prdimers using the nucleotides provided in the
reaction and the genomic DNA as template.
o If the process of replication of DNA is repeated many times,segment of DNA can be
amplified to approximately billion times.
o Such repeated amplification is achieved by the use of a thermostable DNA polymerase
o (Taq polymerase, enzyme obtained from bacteria called ThermUS AQUatICUS )
4. Insertion of Recombinant DNA into the Host Cell / Organism:
 In this step, the recombinant DNA is introduced into a recipient host cell. This process is
termed as Transformation.
 Once the recombinant DNA is inserted into the host cell, it gets multiplied and is expressed in
the form of the desired product under optimal conditions.
 There are several methods of introducing the ligated DNA into recipient cells.
5. Obtaining the Foreign Gene Product:
 The recombinant cells can be multiplied in large scale using a continuous culture system.
 Once the foreign DNA is inserted in to host, it is multiplied and ultimately desirable
protein is produced.
 For the production of the desired protein, the gene encodes for it needs to be expressed.
BIOREACTORS
 Large scale production of desired proteins can be achieved by using bioreactors.
 In a bioreactor about 100 to1000 litres of cultures are processed.
 A bioreactor is a large culture vessel in which raw materials are biologically converted into
specific products.
7 Prepared by Ismail Parambath,KKM Govt.HSS,Orkatteri,Kozhikkode

Downloaded from www.hssreporter.com

  The culture is done by using microbial , plant or human cells.
 In a bioreactor availability of optimum temperature , pH, substrate, salts, vitamins and
oxygen are provided for culture.

Stirred-tank reactor:
It 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.
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
6. Downstream Processing:
 The product obtained in a culture or a bioreactor is not in pure form.
 The downstream processing involves those processes and methods that are responsible for the
separation and purification of the desired product.
 The desired product has to be separated from the culture and formulated with suitable
preservatives.

***********************

8 Prepared by Ismail Parambath,KKM Govt.HSS,Orkatteri,Kozhikkode

Downloaded from www.hssreporter.com