ONE SHOT BIOTECHNOLOGY

2-3 Questions will be from here

INTRODUCTION
UNITS TO BIOTECHNOLOGY

TISSUE CULTURE

PROTOPLAST FUSION

BIOREACTORS

UPSTREAM AND DOWNSTREAM PROCESSING

ENZYME IMMOBILIZATION

BLOTTING TECHNIQUES
UNIT 1: INTRODUCTION TO BIOTECHNOLOGY
INTRODUCTION TO BIOTECHNOLOGY
•Term biotechnology was introduced in 1917 by a Hungarian engineer, Karl Ereky.

•Ereky defined biotechnology as 'all lines of work by which products are produced
from raw materials
with the aid of living things

•Another Definition - The use of microbial, animal or plant cells or enzymes to DOLLY
synthesize, breakdown or transform materials.

•Father of Biotechnology is Louis Pasteur

Humulin (Human Insulin) is

INTRODUCTION TO BIOTECHNOLOGY
YEAR IMPORTANCE

1865 Inheritance of Genetic Traits by Gregor Mendel

1876 Louis Pasteur identified role of microbes in fermentation

1917 Karl Ereky coined the term Biotechnology

1928 Discovery of Penicillin by Alexander Fleming

1944 Identification of DNA as the genetic material (Avery, Macleod and McCarty)

1953 Determination of DNA Material by Watson and Crick

1961 Lac Operon Model proposed by Jacob and Monod

INTRODUCTION TO BIOTECHNOLOGY
1973 Establishment of recombinant DNA technology by Boyer and Gohe

1975 Production of Monoclonal Antibodies by Kohler and Milstein

1976 Sanger and Gilbert developed techniques for DNA Sequencing

1978 Production of Human Insulin in [Link]

1982 U.S.-approved Humulin (human insulin), first pharmaceutical product of rDNA technology, for human use

1988 Development of Polymerase Chain Reaction(PCR)

1990 Launch of Human Genome Project

1997 First Mammalian Sheep, Dolly, was developed by Nuclear Cloning

UNIT 2 :TISSUE CULTURE
What to study in Tissue Culture?

✓ Definition of PTC

✓ Definitions – Explant, Meristem, differentiation, dedifferentiation,

Totipotency , Callus

✓ Types of Culture – Callus Culture, Suspension Culture, Organ Culture,

Protoplast Culture

✓ Subculturing
SPECIAL POINTS
Hairy root Culture
✓ It is a type of organ culture.

✓ In such culture, bacterium named Agrobacterium rhizogenes is inoculated with explant.

✓ Agrobacterium rhizogenes contains root inducing (Ri) plasmid which causes hairy root culture.

✓ The hairy roots so produced have the secondary metabolites.

✓ Ti Plasmid - Found in Agrobacterium tumefaciens. It is responsible for causing crown gall disease in plants by transferring T-
DNA into the plant genome, leading to tumor formation.
SECONDARY METABOLITES
✓ Colored secondary metabolites are identified via Visual Analysis. Radioimmunoassay is most common method.

✓ For metabolites stored within the vacuole of the cells, Permeabilizing agents such as Dimethyl Sulfoxide (DMSO) is used.
This agent disrupts the membranes of cell and cause the release of the metabolites.
IMPORTANT TABLE
Hemocytometer Manual Counting of Cell

(Cell Counting Chamber)

Osmometer Determine osmolality

Phase Contrast Microscopy Check Viability of Cells

[

Nitrogen Freezer Cryopreservation of Cell Lines and Storage

UNIT 3 :PROTOPLAST FUSION
PROTOPLAST FUSION (SOMATIC HYBRIDIZATION)
✓ Protoplasts are naked plant cells without cell wall. They do have plasma membrane.

✓ Protoplasts of different species can be fused to generate hybrid and this is called somatic hybridization/protoplast
fusion.

✓ Protoplast term was coined by Hanstein in 1880 and first isolation was made by Klercker(1892)

✓ Most common source of protoplast is Mesophyll tissue of leaves of young plants.

PROTOPLAST FUSION (SOMATIC HYBRIDIZATION)
1. Isolation of Protoplasts
Mechanical Method
✓ Rarely used now since it is slow process and yield is very low.
✓ Plasmolysis is carried out to release the protoplast from the cell wall.

Enzymatic Method
✓ Preferred since yield is more
✓ Enzymes used are cellulase, pectinase(macerozyme) and hemicellulase as cell wall is made up of cellulose, pectin
and hemicellulose. Cellulase and pectinases are mostly used.
PROTOPLAST FUSION (SOMATIC HYBRIDIZATION)
2. PURIFICATION
✓ Protoplasts are purified by filtration and followed by centrifugation.
✓ Purification is necessary as sometimes broken protoplasts and undigested cells are there.
PROTOPLAST FUSION (SOMATIC HYBRIDIZATION)
3. Viability of Protoplasts
1. Evan’s Blue Staining
Dilute solution of Evan’s Blue (0.025%w/v) is used to stain dead or damaged cells. Viable cells are unstained.

2. Fluorescein Diacetate Method (FDA Method)

✓ Esterase enzymes of living cells cleave FDA.
✓ Polar portion of fluorescein emits green fluorescence under UV light.
✓ This method is useful to detect living cells as fluorescein accumulates only in the living cells.
✓ Concentration used in 0.01%

3. Phenosafranine Strain – It is taken up by dead protoplasts which turn red whereas viable remains constant.

4. Calcofluor White binds to newly formed cells and emit fluorescence.

PROTOPLAST FUSION (SOMATIC HYBRIDIZATION)
[Link] Methods for Protoplast
1. Feeder layer technique
✓ It is preferred for low density protoplast culture.
✓ In this method, X-rays are hit on the protoplast cell suspension.

2. Co-culture
In such method, protoplasts of two different species(one slow growing and one fast growing) are cultured.

3. Microdrop Culture
PROTOPLAST FUSION (SOMATIC HYBRIDIZATION)
Fusion Methods(GPAT 2013)

1. Mechanical Fusion - Protoplasts are mechanically fused which

can sometimes cause damage.

FUSION
METHODS

SPONTANEOUS MECHANICAL INDUCED

PROTOPLAST FUSION (SOMATIC HYBRIDIZATION)
2 Spontaneous Fusion
•It occurs naturally when protoplasts of two species adjoin to form homokaryons.

•Fused cells have high number of nuclei (2-40) due to expansion and subsequent coalescence of plasmodermal connections between
cells

•Disadvantage – Such Fusion does not generate whole plant.

•Homokaryon- A cell that has multiple, identical nuclei in

common cytoplasm.
•Heterokaryon - A cell that has multiple, genetically distinct
nuclei in common cytoplasm.
•Plasmodermal connections -These are cytoplasmic bridges that
allow materials to move between two cells.
•Coalescence–It means process by which two cells come in
contact with each other.
•Somaclonal variations – Mutations/Variations that occur during
plant tissue culture
PROTOPLAST FUSION (SOMATIC HYBRIDIZATION)
3. Induced Fusion
Fusogen (Fusion inducing agents) are used.

(SPLEND -C)
Remember Bold Fusogens
S – Dextran Sulfate

P - Polyethylene glycol (PEG) and Polyvinyl alcohol (PVA)

L – Lyzozyme

E – Esters and Fatty Acids/ Electrofusion

N - NaNO3

D – Dextran

C - Ca2+ at High pH
NaNO3 Use of 5.5% NaNO3 in 10% sucrose solution

Ca2+ at High pH 0.4M Mannitol containing 0.05 M CaCl2 at pH = 10.5 at room temperature.

PEG Method of choice due to low toxicity, non-specificity.

Electrofusion ✓ Use of Electric field to fuse the protoplasts.

✓ Electric field is created by electrodes.

✓ This method shows no toxicity but cost is high.

UNIT 4 : BIOREACTORS
INTRODUCTION

✓ Fermentation is a biochemical process that produces chemical changes in organic substances by the action of
enzymes.

✓ There is an absence of oxygen in fermentation. But, currently in industrial fermentation, we are using oxygen also.

✓ First fermented product was alcohol.

TYPES OF FERMENTATION
Type Product Special Points
Lactic Acid is produced from the pyruvate
produced in glycolysis.
Lactic Acid Fermentation 1. Lactobacillus produce curd from
milk (Concept from Biochemistry)
2. Sauerkraut (Type of Food)
1. In such fermentation, end product is
alcohol and CO2.
Alcohol Fermentation/Ethanol Wine, Beer, Biofuel, bread and baking
Fermentation process 2. Yeast is most commonly used.

3. Enzymes involved are pyruvic acid

decarboxylase and alcohol dehydrogenase

Acetic Acid Fermentation Vinegar, polymer 1. Yeast is most commonly used.

2. Other used – Acetobacter bacteria

3. Aerobic Process

Butyric Acid Fermentation Used in flavouring agents and Tobacco 1. Such fermentation is only done by
Processing obligate anaerobic bacteria (Clostridium)

2. Oxidoreductase enzyme is involved

STRUCTURE
BASICS OF BIOTECHNOLOGY
•Bioreactors are made up of stainless steel.

•Size of bioreactors –Small ( 1-20L) and Large (>450L)

•Shape - Cylindrical

•Temperature : 150-180° C

•Pressure : 377-412 kPa

 COMPONENT AND WORKING OF BIOREACTOR
REACTION VESSEL •It is surrounded by Jacket and have sparger at bottom.(GPAT 2015)

•Through sparger, air (and gases such as CO2 and NH3) are introduced from bottom under pressure.

•Impeller breaks down the bubbles generated by spargers and dispersed it throughout medium in an uniform manner.
Speed of impellers determines stirring rate.

•Larger the bioreactor, lesser the speed of impeller.

Aeration •It is necessary for exchange of gases(Supply O2 to organisms and remove CO2)

•O2 is stored in compressed form and it is supplied from bottom(in form of bubbles)

•O2 rises to top and accumulate in headspace and finally pass out via air outlet.

•Aeration rate is in range of 0.25-1.25vvm (Volume of air/volume of liquid/minute)

Foam •Protein present in bioreactor during aeration produces froth or foam which accumulates in headspace.

•Antifoam chemicals and Mechanical Foam Control Devices are used which burst the foam bubbles. Example – Mineral
Oils based on silicone or vegetable oils.
TYPES OF BIOREACTORS
Continuous Stirred Tank Bioreactor ✓ A well-mixed bioreactor equipped with an impeller for continuous
stirring, ensuring uniform conditions.

✓ Suitable for processes requiring constant mixing and precise control over
environmental factors like pH and temperature.

✓ Name of impellers – Rustom Disc, Marine propeller

Airlift Bioreactors ✓ Uses air to circulate and mix contents without mechanical stirring.

✓ Suitable for delicate cells as it reduces shear stress.

Fluidized bed bioreactor ✓ Contains small particles suspended by upward fluid flow, increasing
surface area for reaction.

✓ Ideal for processes requiring high mass transfer.

TYPES OF BIOREACTORS
Packed Bed Bioreactors ✓ Features immobilized cells or enzymes packed in a column, with liquid
medium circulating through.

✓ Ideal for continuous product recovery.

Photobioreactors ✓ Uses light as an energy source for culturing photosynthetic organisms

such as algae.

✓ Typically designed with transparent surfaces to maximize light exposure.

✓ Beta-carotene is produced by such type of bioreactor.

Bubble Column Bioreactors ✓ A simple design where gas is sparged at the bottom to create bubbles,
ensuring effective gas-liquid contact.

✓ Suitable for aerobic cultures with low viscosity.

STERILIZATION IN FERMENTATION
These are mainly contaminated by vegetative cells and spores. Methods of sterilization are:-

Method of Sterilization Special Points

Heat 1. Most Common

2. Vegetative cells are killed at lower temperature (60° C in

5-10 minutes) and spores are killed are higher
temperature

3. (80°C in 15-20 mins)

Physical Methods 1. Filtration (most common)
2. Centrifugation
3. Adsorption (on charcoal)
#Physical Methods are used in case of heat – labile
constituents such as vitamins

Chemical Methods [Link]

2. Radiations such as UV Rays, Gamma Rays and X Rays.

STERILIZATION IN FERMENTATION
Filtration is most commonly used. Two Types of Filters are used

Method of Sterilization Special Points

Depth Filter ✓ It contains glass wool which traps the particles and
remove them.
✓ Glass wool is sterilized by steam distillation but steam
distillation shrinks and solidifies the wool on repeated use

Membrane Cartridge Filter ✓ It contains removable pleated filters made of cellulose

ester, nylon or polysulfone.
✓ It is small and more used.
SPECIAL POINTS
•Sensors(automated) are installed to continuously monitor the variables such as pH, temperature, O2. Optimum
temperature is required. Low temperature reduces product formation whereas high affect the microbe organism.

.
•20% of space of bioreactor is vacant at top known as Headspace whereas 80% is known as working volume.

•Cleaning of Bioreactor is done by High-Pressure water jets from holes in reaction vessel.

•Hydrostatic pressure of 0.2-0.5bar is used. Pressure influences solubility of O2 and [Link] varies according to type
of fermentation.

•Methanol is cheapest substrate for fermentation and ethanol is used for acetic acid production.

•End- capping is a process used to modify the ends of a biopolymer, such as DNA or RNA, to prevent degradation and
improve stability.
SPECIAL POINTS
Feature How To Measure

pH pH electrodes (Glass and Reference

Electrode)

O2 and CO2 1. Zirconium dioxide or zirconia

lambda sensors are used.
[Link] and gas permeable
Membranes.

N2 , NH3 , Ethanol Mass Spectroscopy

UNIT 5 : UPSTREAM & DOWNSTREAM
PROCESSING
INTRODUCTION
Upstream processing is combination of tasks in the starting stages of the fermentation process which includes:-
INTRODUCTION

Upstream Processing

Development of
Nutrient Development Inoculation Cell Culture
Microbes

Batch Culture
Isolation
Refrigeration Fed-Batch Culture
Screening
Done in Cognosy Freezing Semi-Continuous
Preservation
Lyophilization Culture
Genetic Improvement
Continuous Culture
DEVELOPMENT OF MICROBES
DEVELOPMENT OF MICROBES
INOCULATION
• It is process of adding a population of microorganisms or cells in the medium.

• Population which is added is called inoculum.

• Inoculum is taken from stock culture.

•Stock Culture – Culture of microorganism is stored or maintained so that microorganism remains in the viable state.

1. Preservation of Inoculum

Preservation is important so that inoculum remains viable as long as possible without any cell division.
Methods used are:-

Refrigeration Temperature = 2-6°C

Simple but contamination can happen easily

Freezing Temperature = -18 - -80° C

-196° C with Liquid Nitrogen

Lyophilization (Freeze Drying) Best Method

INOCULATION
2. Build-up of Inoculum

•It means when you revive preserved inoculum for use.

•The time taken by build-up and technique used depend upon method of preservation.

Method Time to Revive

Refrigeration 6-24 Hours (For Bacteria)

Freezing 6-48 Hours (For Bacteria)

Lyophilization (Freeze Drying) 4-10 Days (For all organisms)

•After revival, inoculum is suspended in Tween 80 and then transferred to bioreactor.

•Concentration of inoculum taken for bacteria is 0.2 to 3% and 5-10% for fungi.
CULTURE MEDIUM
CULTURE FEATURES
Batch Culture (Closed System) In such culture, nutrient medium/substrate is added in starting only.
No fresh medium is added afterwards.

Fed-Batch Culture In such culture, substrate is added in increments at regular intervals.

Yield is comparatively more (25% to 1000% times) as compared to Batch
Culture.

Semi-continuous Culture In such portion, portion of medium is removed and replaced by fresh
medium.
It can be done at repeated intervals.
Yield is greater than batch and fed-batch culture.
Technical difficulties and contamination on repeated addition of medium
are limitations.

Continuous Culture (Open System) In such portion, medium is removed and replaced by fresh medium such
that rate of removal and addition of medium is same.
Place it in table form or Smart Art Form
DOWNSTREAM PROCESSING
✓ It is defined as extraction and purification of product from fermentation.

✓ It is also known as product recovery.

✓ Fermentation and Downstream processing are considered as integrated system.

✓ Lesser the number of steps in DSP, more is the yield.

✓ There are two types of metabolites obtained from DSP:-

A. Intracellular Metabolites – Metabolites present within the cell(Vitamins and enzymes)
B. Extracellular Metabolites - Metabolites present outside the cell (Antibiotics, amino acids, alcohol)
CULTURE MEDIUM
DOWNSTREAM PROCESSING - STEPS
Harvesting

Release of
Intracellular
Products

Concentration

Purification

Formulation
HARVESTING
Solid – Liquid Separation (Harvesting)
In this step, cells are separated from the culture medium. Various methods are there:-

Flotation ✓ Density based separation method

✓ There is the formation of bubbles
Flocculation ✓ Cells form large aggregates to settle down for
removal.
✓ Flocculating agents are used such as
inorganic salts
Filtration(Most common) Several filters are used such as

Mnemonics Mania (MARD)

M – Membrane Filters

A - Absolute filters

R – Rotary Drum Filter

D – Depth Filter

Centrifugation ✓ Density based separation method.

✓ There is a difference between the density of
cells and medium.
CELL DISRUPTION
Release of Intracellular Products (Cell Disruption)
Intracellular products are released by Cell disruption. There are mainly three types of Cell disruption methods:-

Cell Disruption

Physical Methods Enzymatic Methods Chemical Methods

Osmotic Shock
Alkalies
Thermolysis (Heat Shock)
Lysozyme Organic Solvents
High Pressure
Detergents
Homogenization
Impingement
CELL DISRUPTION – PHYSICAL METHOD
Osmotic Shock Use of water and sucrose
Thermolysis (Heat Shock) Cause breakage of cells due to heat shock.

Used for heat stable substances only.

High Pressure Homogenization Cause breakage of cells due to high pressure applied in narrow
orifice.

Impingement (Hit or Strike) ✓ Stream of cells with high pressure and velocity are made to
hit a stationery object or another stream of cells. This
causes cell disruption.
✓ Device made on this principle is Microfluidizer.
✓ It can disrupt cells even at low concentration.
CELL DISRUPTION – CHEMICAL AND ENZYMATIC METHOD
Treatment with Alkalies Example – Sodium Hydroxide
Treatment with Organic Solvents Example – Ethanol, Methanol, Toluene

These dissolve membrane phospholipids and create pores in

membrane.

Treatment with Detergents Cationic – Cetyl trimethyl ammonium bromide (CTAB)

Anionic – Sodium Lauryl Sulfate (SLS)

These denature membrane proteins and break the cells.

 CONCENTRATION
✓ Now, filtrate is free from medium but it contains huge amount of water.
✓ Therefore, water has to be removed.
✓ This is called concentration.
✓ There are various ways to concentrate.
Evaporation In evaporators, you have a heating device which supply steam to evaporate water.

Liquid – Liquid Extraction It is achieved by transferring the desired product from one liquid phase to another phase.

Efficiency depends upon the partition coefficient.

Membrane Filtration In this method, we use permeable membrane that selectively retains particles larger than pore size and smaller particles
pass through membrane.

Membrane is made up of:-

A. Polymeric materials –Polyether sulfone (PES) and Polyvinyl difluoride

B. Microfilters and ultrafilters made of ceramics and steel

 CONCENTRATION
Precipitation Used mainly for macromolecules like proteins.

Commonly used salt - Ammonium Sulfate

Adsorption Cellulose based adsorbents and PAM are used

P – Polystyrene

A – Acrylate

M - Methacrylate
PURIFICATION AND FORMULATION
•Chromatography is used.
•Types of chromatography used are:-

Gel – Permeation Chromatography

Affinity Chromatography

Ion- Exchange Chromatography

It is about maintaining the stability of product during storage and distribution.

Formulation is done in following manner:-

For Low Molecular Weight Compound Concentration is done

For Small molecules (like antibiotics) Crystallization is done
For Macromolecules Stabilizers are added.

Example – Sugars (Glucose), Salts (NaCl), Polymers (PEG).

Polyhydric alcohols
UNIT 5 : ENZYME IMMOBILZATION
BASICS
Some Special Points about Enzyme
✓ Enzyme are non-toxic and biodegradable in nature.

✓ First enzyme produced was Takdiastase in 1896 in USA.

✓ Fungi (60%) contributes maximum to enzyme production.

✓ L-amino acid acylase was the first enzyme to be immobilized in 1969.

Microbial sources are preferred over animal sources for enzymes because:-
1. Quantities produced from animal sources is in less quantity.
2. Isolation and purification of enzymes is difficult from animal sources.
3. Extraction from bovine sources leads to high risk of contamination causing Bovine Spongiform Encephalopathy(BSE), a
type of disease.
ENZYME IMMOBILZATION
✓ Immobilization of enzymes refer to confining the enzymes in or an inert support medium for their stability and
functional reuse.
✓ It is done because free enzymes in solutions are not stable and cannot be reused therefore, they are wasted.
ENZYME IMMOBILZATION
ENZYME IMMOBILZATION
Advantages Disadvantages

Immobilized enzymes retain their structural Loss of biological activity during process can happen.
conformation necessary for catalysis.

They are stable and efficient in function. Expensive

They can be reused and hence cost-effective

Products are enzyme-free

UNIT 6 : BLOTTING TECHNIQUES
ISOLATION AND PURIFICATION OF DNA
Type of Cell Treatment Required

Bacterial Cell Lysozyme + Ethylenediamine tetraacetate (EDTA) followed

by Sodium dodecyl Sulphate (SDS)

Animal Cell SDS

Plant Cell Freeze + Use of Mortar &Pestle (Harsh treatment is done
because cell wall is strong)

INDIRECT METHOD
1. Centrifugation - helps in removing the cell debris.
PURIFICATION 2. RNase – Degrade RNA
3. Ethanol – Helps to precipitate DNA.

DIRECT METHOD
TRADITIONAL
DIRECT WAY We add detergent called Cetyltrimethylammonium(CTAB) which makes
WAY
insoluble complex with nucleic acids.
ISOLATION AND PURIFICATION OF DNA
Silica-Based DNA Extraction with Guanidinium Thiocyanate : (NOT IMPORTANT)
✓ We bind DNA with silica particles in a presence of denaturing agent called guanidinium thiocyanate.

✓ Then we do centrifugation to get DNA.

BLOTTING TECHNIQUES
Southern Blotting 1. It was developed by Ed Southern in 1975.

2. It is used for identification of DNA.

3. We use nitrocellulose paper or nylon membrane.

Northern Blotting 1. It is used for identification of RNA.

2. For RNA molecules, we use modified nitrocellulose paper or nylon

membranes as normal is not able to bind to the RNA.

Western Blotting 1. It is used for identification of proteins.

2. Proteins are detected by specific protein-ligand interactions.

3. In western blotting, we use antibodies or lectins.

Dot Blotting (Modification of southern and 1. It is used for identification of both DNA and RNA.
northern blotting)
SPECIAL POINTS FOR NIPER
Purification of mRNA – It is done by affinity chromatography using (dT)-cellulose

Purification of Plasmid is done by - Isopycnic centrifugation method and Birnboim and Doly Method