Study Notes BIOTECHNOLOGY – Principles and Processes

BIOTECHNOLOGY
Definition:
 Use of live organisms or enzymes from organisms to produce products or processes
useful to human.
 The integration of natural science and organisms, cells, parts thereof and molecular
analogues for products and services

Two principles of Biotechnology: which gave birth to field of modern biotechnology –

1. Genetic Engineering:
a. ability to cut, manipulate and join DNA and RNA molecules
b. ability to introduce foreign DNA into a host cell and change its phenotype
2. Maintenance of sterile environment:
a. Ability to maintain a germ free / microbial contamination free environment for
growth of desired microorganisms
b. Ability to produce biotechnological products in large scale in pure form.

Construction of first recombinant DNA:

 First recombinant DNA was constructed by Stanley Cohen and Herbert Boyer in the year
1972
 An antibiotic resistance gene was isolated from a plasmid by cutting it with molecular
scissors – REE.
 The cut piece of DNA was linked with a plasmid of Salmonella typhimurium using DNA
ligase enzyme.
 These recombinant plasmid acts as a vector to transfer a foreign DNA into a cell.

TOOLS and TECHNIQUES used in BIOTECHNOLOGY PROCESSES

A. RESTRICTION ENDONUCLEASE ENZYME (REE)

 For isolating the desired gene / DNA fragment from the entire DNA, a DNA cutting tool is
required. A nuclease enzyme acts as a DNA cutting tool.
 Nuclease – any enzyme which can cut the DNA (nucleic acid) at a random site is called a
nuclease enzyme
 It is of two types:
o Endonuclease – a nuclease enzyme which cut the DNA within the molecule
o Exonuclease – a nuclease enzyme which cut the DNA from its 3’ or 5’end.
o Both the above can cut the DNA at any random site.
 Restriction Endonuclease Enzyme (REE) – it is an endonuclease enzyme which recognises
a specific sequence known as Recognition sequence and cut the DNA based on it.
o Hind II was the first restriction endonuclease enzyme isolated
o They are obtained from various strains of bacteria
o More than 200 enzymes have been isolated from more than 1000 strains of bacteria
o They are of three types: Type-I, Type-II and Type-III
 Type I and Type III recognise recognition sequence but cuts the DNA some
base pairs away from it
 Type I enzyme cuts the DNA within the recognition sequence
If REE are enzymes present in bacteria, why does it not act on the naked genetic material
of bacteria? What is its function?
Restriction Modification System
Restriction system: REE in bacterial cytoplasm, acts on the phage DNA which infect the
bacteria and restricts its activity, hence the name.

Notes prepared by MRINAL CHOUDHURY Page 1 of 10

 Study Notes BIOTECHNOLOGY – Principles and Processes

Modification system: Methylase enzyme carries out methylation of bacterial DNA and
hence REE does not act on it?

 Naming the RE Enzymes:

o First three letters are derived from the organisms from where it is isolated
o Fourth letter is derived from the type of plasmid or strain of the bacteria
o Roman numeral means the serial order of various enzymes obtained from the same
organism

 Recognition sequence – the specific DNA sequence recognized by Restriction endonuclease

enzyme is known as Recognition sequence.
o These sequences are palindromic sequence – a sequence which reads the same from
both the directions when the polarity is same.
o E.g. Recognition sequence of Eco RI is 5’GAATTC3’
3’CTTAAG5’
 REE recognises the specific recognition sequence and cuts the DNA within the sequence by
breaking the sugar phosphate phosphodiester bonds in two different ways:

 When two DNA molecules are cut by similar REE, similar sticky ends are produced which
are complementary and therefore easily attach to each other by hydrogen bonds. Later DNA
ligase produce the phosphodiester bond between the two molecules, forming the recombinant
DNA.

Notes prepared by MRINAL CHOUDHURY Page 2 of 10

 Study Notes BIOTECHNOLOGY – Principles and Processes

B. ELECTROPHORESIS

 ‘Electro’ means ‘electric field’ and ‘phoresis’ means ‘movement’.

 It is a process of separating DNA fragments based on their size or molecular weight.
 RNA or proteins can also be separated by same principle.
Procedure (process flow chart)
Agarose gel is made along with ethidium bromide(added in the molten state)
 Agarose gel has minute pores through which DNA fragments can move
 Ethidium bromide can help in visualizing the DNA in the gel later
↓
Gel has multiple wells where the DNA sample is loaded
↓
The electrophoretic unit is connected to a power supply in such a way that DNA loaded wells are
towards negative electrode

 Agarose gel has minute pores through which DNA fragments move under the electric field.
 The smallest fragments move maximum distance in the gel, while the larger fragments move
lesser distance and hence all the fragments are separated on the basis of their size.

Notes prepared by MRINAL CHOUDHURY Page 3 of 10

 Study Notes BIOTECHNOLOGY – Principles and Processes

 Visualization of DNA bands –

o Visualization of DNA bands in the gel can be done by staining the gel with stains
(commonly used Bromophenol Blue) and destaining the excess stain multiple times.
o The process is made simpler by using Ethidium Bromide (Etbr).
 Etbr is added to the gel in its molten state.
 When DNA is separated in the gel, Etbr binds with the DNA forms a
complex.
 The same complex fluoresces under UV light and can be observed as orange
colored DNA bands.

 Band identification using DNA LADDER

o A DNA ladder is a mixture of different DNA fragments with their known fragment
size
o By comparing the separated fragments with the DNA ladder, the size of the unknown
fragment is determined or the required DNA band is identified

 Gel Elution – the specific DNA band of identified length is cut out from the gel and the
DNA fragments are purified from it.

C. POLYMERASE CHAIN REACTION (PCR)

 It is a process of DNA amplification.

 If the amount of DNA for an experiment of study is present in very less quantity, they can be
amplified to increase the quantity.
 In 1985, Kary Mullis discovered this technique.
 Requirements:
o The target DNA molecule to be amplified
o RNA Primer – artificially synthesized, short oligoribonucleotides of desired sequence

Notes prepared by MRINAL CHOUDHURY Page 4 of 10

 Study Notes BIOTECHNOLOGY – Principles and Processes

o Taq polymerase – it is a DNA polymerase obtained from bacteria Thermus aquaticus

found in hot springs. It can withstand very high temperature, upto 90OC and be active.
o dNTPs – free nucleoside triphosphates for polymerization
 Process: the process PCR is divided into three steps
o Denaturation: separation of two strands of target DNA
o Annealing: joining the primers to the 3’ end of each strand
o Extension: polymerization of nucleotides to form new strands

The target DNA / gene is isolated from source

Denaturation ↓ temp ~90OC

The two strands get separated

Annealing ↓ primers are added,
temp ~ 50-70OC(depends on primer)

Primer binds to the 3’ end of the two strands

Extension ↓ taq polymerase and dNTPs added
Temp ~ 72OC

Polymerisation of nucleotides complementary to the template strands will take place,

making two new molecules of DNA
↓
The cycle of above three steps is repeated many times to produce large number of molecules.
 Since in every cycle, number of DNA molecules keep doubling, a large number of molecules
are synthesized in a short period of time.

D. CLONING VECTOR

 A vector is a DNA molecule which help in the transfer of foreign DNA into a host, which is
otherwise not accepted by the host.
 The foreign DNA may be recombined to a vector to make a recombinant DNA molecule
which is transformed into the host cell.
 The cloning vectors are most commonly a Bacteriophage genome or a Plasmid DNA

Bacteriophage Vector Plasmid Vector

 Bacteriophage are very efficient in  It is a non-chromosomal DNA of a
infecting a bacteria and multiply inside bacteria which carry some important
the host cell but non-essential genes.

Notes prepared by MRINAL CHOUDHURY Page 5 of 10

 Study Notes BIOTECHNOLOGY – Principles and Processes

 So a foreign DNA linked to its genome  They may be present in single copy or
can easily multiply the copies of foreign multiple copies in a bacterial cell.
DNA o Single copy plasmid
o Multi copy plasmid

 These vectors are either used directly or now-a-days engineered to incorporate desirable
characters to which foreign DNA can be linked.
 A good vector must have following features:
o Origin of Replication (ori):
 Ori sequence is the site of origin of replication
 It also regulates the copy number of a plasmid
 A foreign DNA must be linked to the ori sequence, else the foreign DNA will
not replicate and be maintained in the cell.
 Linking with ori sequence which allow high copy number is necessary if
multiplication of the foreign DNA is desired.
o Selectable Marker:
 A Marker gene is a gene that helps in identifying a specific sequence in the
DNA.
 Selectable marker gene help to identify the successful recombinants and
transformants in genetic engineering experiments
 Selection is done by insertional inactivation of a marker gene
 Marker genes are of two types:
 Antibiotic resistance gene
 β galactosidase gene
o Cloning sites:
 These are recognition sequences for various restriction endonuclease enzyme.
 The plasmid may be cut open at any of these sites by digesting with the
respective enzyme and clone the foreign gene into it.
 It is preferable to have “unique cloning sites for multiple enzymes”.
o Ti plasmid of Agrobacterium tumifaciens
 Agrobacterium tumifaciens has a natural ability to infect plant cells and
introduce its Ti plasmid into it.
 Ti plasmid is Tumor Inducing plasmid and causes a disease in plants known
as crown gall disease.
 Ti plasmid has two distinct type of genes in it –
 Vir genes: virulence genes which are responsible for causing the
disease. They are removed by the process known as Disarming, so that
it looses its ability to cause tumor
 T-DNA: Transfer DNA is responsible for Ti plasmid into plant cells
successfully. Disarming of Ti plasmid does not disturb its infectivity.
 After disarming, the foreign DNA can be cloned to the Ti plasmid and is
transformed into a Agrobacterium.
 Then it will naturally introduce the foreign DNA into a plant cells if a plant
cell is exposed to infection by such recombinant and transformed bacteria.
o Retrovirus – vector for cloning foreign DNA into animal cells.
 Retroviruses are very efficient in infecting animal cells and introducing its
genetic material into animal host cells.
 However, they are also responsible very causing severe diseases in human.

Notes prepared by MRINAL CHOUDHURY Page 6 of 10

 Study Notes BIOTECHNOLOGY – Principles and Processes

 So they are disarmed and foreign DNA is cloned with retroviral genetic
material.
 It is then able to naturally transform the animal cells with the foreign DNA.
E. COMPETENT HOST

 Since DNA is hydrophilic, it does not pass through the cell membranes spontaneously.
 Therefore a recombinant DNA molecule will not be taken up by the bacterial cell easily.
 Therefore, the host cell needs to be made competent, so that it can take up the recombinant
DNA.
1. Microporation – for making competent Bacterial Cells
 Bacterial cells are mixed with Ca2+ ions. Ions increase the efficiency of DNA entry into
the cell by inducing the pore formation in the cell wall.
 Cells are incubated on ice, then transferred briefly at 42OC and then putting them back on
ice. It is called Heat Shock.
 Heat shock creates transient pores in the cell wall of the bacteria and the DNA enters
inside.
 Instead of Heat shock, Electric shock also can be provided through two electrodes.

2. Microinjection – for making competent Animal cells

 The cell to be transformed is hold
by a micropipette
 The foreign DNA is taken in a
microinjection
 With the help of a microneedle,
the DNA fragment is directly
introduced within nucleus of the
host cell

3. Gene Gun / Biolistic – for making competent Plant cells

 The foreign DNA is encoded into an inert
substance such as gold or tungsten
 The encoded DNA is bombarded into plant cells
with high velocity
 Due to high velocity, DNA fragments enter the
plant cells.

Notes prepared by MRINAL CHOUDHURY Page 7 of 10

 Study Notes BIOTECHNOLOGY – Principles and Processes

PROCESS of RECOMBINANT DNA TECHNOLOGY

Step – I: Isolation of DNA

Treatment with enzymes to break open the cell
Bacterial cell Plant cell Fungal cell
Lysozyme Cellulase, Pectinase, etc Chitinase
↓
Purification of DNA from other contaminants such as Proteins and RNAs by treating with
Proteases and Rinonucleases
↓
Centrifugation for separating the DNA from all the debris
↓
Chilled ethanol precipitation of DNA

Step – II – Cutting of DNA to isolate specific gene of interest / foreign gene / alien gene

The source DNA is subjected to digestion by Restriction Endonuclease Enzyme

↓
REE cuts the DNA at specific recognition sites flanking the gene of interest to cut out the same,
more fragments of DNA may also be present

Plasmid DNA is also digested with the same restriction endonuclease enzyme, so that similar
single stranded regions at the ends (sticky ends) are formed

Step – III – Separation of gene of interest / foreign gene from the mixture of other DNA
fragments
Electrophoresis is carried out to study the progression of restriction enzyme digestion
↓
Successful digestion of source DNA with REE will produce more bands in the gel
↓
The DNA band containing the desired gene of interest is identified by comparing with DNA
ladder and isolated by gel elution

Step – IV – Amplification of DNA (optional)

If the required gene of interest is very less in quantity, the same may be increased in quantity by
applying Polymerase Chain Reaction (PCR)
↓
The amplified gene of interest is again purified from PCR products by electrophoresis

Step – V – Recombination
The gene of interest and opened Plasmid DNA are incubated together
↓
The joining of the two produces recombinant DNA molecules known as Recombinants

Step – VI – Transformation
The recombinants are incubated with competent host cells
↓
Entry of recombinant DNA molecules into the host cells produce successful transformants

Step – VI – Selection of Recombinants and Transformants

Notes prepared by MRINAL CHOUDHURY Page 8 of 10

 Study Notes BIOTECHNOLOGY – Principles and Processes

Since recombination and transformation cannot be observed directly, successful recombinants

and transformants must be selected out from non-recombinants and non-transformants
↓
It is done based on Insertional Inactivation of Marker genes
↓
The plasmid contain two marker genes –
 One antibiotic resistance gene helps in selecting transformants from non-transformants
 Other antibiotic resistance gene is inactivated by insertion of gene of interest, which help in
selecting recombinants from non-recombinants
 Since it requires simultaneous plating of hosts in two antibiotic medium plates, it is
cumbersome
 Inactivation of β-galactosidase enzyme by inserting foreign DNA fragment within it makes it
easier to do selection in a single step
↓
The colonies of bacteria containing successful recombinants ad transformants are pure cultured
separately

Step – VII – Obtaining Foreign Gene Product

The desirable gene product is produced in large quantities by growing the transformant bacteria
in large culture vessels known as Bioreactor
Bioreactor
 Since they have large capacity of 100 – 1000ltrs, large volume of culture can be done at a
time for obtaining the gene product
 They provide optimal conditions for the growth of host cells and production of the gene
product
 They are of two basic types :
o Simple Stirred Tank Bioreactor
o Sparged Stirred Tank Bioreactor
 Simple Stirred Tank Bioreactor – parts and functions
o Stirrer / Impeller – for continuous mixing of
medium so that organisms are able to utilize
all the available nutrients
o Foam breaker – removes foam produced at the
top of the broth due to continuous stirring
o pH control – growth of microorganisms may
lead to change in pH of the medium which can
alter the quality of the product, so the same
need to regulated
o Sterile air supplied directly
o Temperature regulator is available for
checking the temperature change during the
growth
o Sample collection port – for collecting sample at regular intervals and check the
growth of organisms and production of product
o Steam supply port – Steam is pumped inside the bioreactor for sterilization of the
reactor
 Sparged Stirred Tank Biorector – differences from simple stirred tank bioreactor

Notes prepared by MRINAL CHOUDHURY Page 9 of 10

 Study Notes BIOTECHNOLOGY – Principles and Processes

o All the parts are same as simple stirred

tank bioreactor except the sterile air
pumping system
o Sterile air is passed through a sparger
 A lot of air bubbles are formed
inside which increase the oxygen
transfer area
 Surface area of the broth also
increases the oxygen transfer

Type of Culture in Bioreactor

 Of culture in the bioreactor may be Batch culture or Continuous culture
Batch Culture Continuous Culture
 When the product is required in lesser  When the product is required in greater
quantity quantity and is needed to be produced very
regularly
 The culture is done in batches, where  The culture is continuous replenished with
fresh medium is inoculated with cells in fresh medium, while used medium is taken
different batches out of the bioreactor
 Bacteria completes its growth cycle,  Bacteria always remains in exponential phase
before the product is purified of its growth

Step – VIII – Downstream Processing

 After the gene product or Recombinant protein is produced in a bioreactor in large scale, it
must undergo Downstream processing, which include various stages of purification.
Multiple rounds of filtration
↓
Purification (by the processes of chromatography)
↓
Formulation : in what form it will be available for usage/consumption
 Tablets
 Capsules
 Syrup
 Injections: IV or IM
 Infusions
 Creams and Jellies
 Powder
 Dermal patches, etc
↓
Clinical Trials: for assessing safety and efficacy of the product
 Animal toxicity testing
 Human clinical trials
↓
Packaging
↓
Marketing

Notes prepared by MRINAL CHOUDHURY Page 10 of 10