Applied Biology – Mod 12-

CSIR NET
Suman Bhattacharjee
Examples of transgenic animals
Transgenic
plants
Marker assisted selection in plants
Traditional vs MAS breeding
MAS- gene level
Gene therapy – Mod 12
Suman Bhattacharjee
 Basic Process of gene therapy
Gene therapy is the insertion, alteration,
or removal of genes within an individual's
cells and biological tissues to treat disease.

It is a technique for correcting defective

genes that are responsible for disease
development.

The most common form of gene therapy

involves the insertion of functional genes
into an unspecified genomic location in
order to replace a mutated gene, but other
forms involve directly correcting the
mutation or modifying normal gene.

Ex-vivo gene therapy

Presently, gene therapies for the following diseases are being developed:
cystic fibrosis
 HIV infection
malignant melanoma
kidney cancer
Gaucher's Disease
breast cancer
and lung cancer

•The medical has contributed to transgenic research that is supported by government funding.

•In 1991, the U.S. government provided $58 million for gene therapy research, with increases of $15-40 million dollars a
year over the following four years.

•With fierce competition over the promise of major medical benefit in addition to huge profits, large pharmaceutical
corporations moved to the forefront of transgenic research.
Ex-vivo gene therapy
 Advantages of Ex vivo Gene therapy

Possibility to expand different cell populations ex vivo

Select cells in which gene transfer has occurred

Avoid possibility of immune response against the vector that might neutralize gene transfer.

Cells treated through this manner include lymphocytes, stem cells of various deviation, keratinocytes.

adenosine deaminase
Adenosine deaminase
(ADA) deficiency is an
inherited disorder that
damages the immune
system and causes
severe combined
immunodeficiency
(SCID).
In vivo gene therapy
Advantages:
This approach appears simpler than ex vivo gene transfer and once optimized can be applied to a vast series of patients with
same disease.

Disadvantages:

The gene might enter cells other than the desired targets, thus causing unwanted effects.

A vector used for gene therapy might be inactivated or in any case elicit an immune response.

Most cells in our body, including cardiomyocytes, neurons and hepatocytes are post-mitotic stage. This limits application of
some vectors.

First, several tissues are difficult to reach or to transduce at a significantly level in vivo.
 Ex vivo and in vivo gene transfer strategies

Hoff C M , Shockley T R JASN 2002;13:S117-S124

©2002 by American Society of Nephrology

 Methods for gene delivery
The gene transfer methodologies that are exploitable by gene therapy can be divided into
four categories.

1. Simple utilization of naked plasmids (circular, covalently closed DNA molecules) or

short regulatory nucleic acids (oligonucleotides, siRNAs and others), not complexed
with other molecules and simply injected in vivo.

2. Facilitation of nucleic acid entry into the cells by physical methods.

3. Transport of nucleic acids into the cells by lipofection.

4. Embedding of nucleic acid sequences within viral genomes, then exploiting the natural
property of viruses to enter target cells at high efficiency.
Virus Vectors used
Antibody mediated HIV
treatment
Microbial growth NET
Suman Bhattacharjee
Bacterial growth curve
Diauxic growth curve
 Generation time calculations
• This progression may be expressed as a function of 2 as shown in the line above. The number of cells (6) present at a
given time may be expressed as

b = 1x2"
• The total number of cells (b) is dependent on the number of generations (n = number of divisions) occurring during a
given time period. Starting with an inoculum containing more than one cell, the number of cells in the population can be
expressed as

b = a x 2"

Solving the equation for n. the number of generations that occurred between the time of inoculation and the time of
sampling is
 Industrial
Microbiology
Introduction
Suman
Bhattacharjee
 Introduction
• Biotechnology can be defined as – “ Application of scientific and engineering principles to
processing of materials by biological agents to provide goods and service.”
• Biological agents, here means, microorganisms, cells, plants and animal cells and enzymes.
• Biotechnology is the culmination of more than 8000 years of human experience using living
organisms and the process of fermentation to make products such as bread, cheese, beer,
and wine.
 Biotechnological process
A typical biotechnological process comprises of the following three steps:
• Step 1: Upstream processing
preparation of liquid medium, separation of particulate and inhibitory chemicals from the medium, sterilization, air
purification etc
• Step 2: Fermentation/Production
conversion of substrates to desired product with the  help of biological agents such as microorganisms
• Step 3: Downstream processing
separation of cells from the fermentation broth, purification and concentration of desired product and waste disposal or
recycle.
 Bioreactors/Fermenters
• The vessel in which fermentation process is carried out
is called a fermenter or a bioreactor.
• The size of a bioreactor depends on the purpose of use.
• Some examples of bioreactors with varying sizes are :
1. Shake flask (100-1000 ml)
2. Laboratory fermenter ( 1-50 lt)
3. Pilot Scale (0.3-10 m^3)
4. Plant Scale (2-500 m^3)
 Requirements of bioreactors
(a) The design and construction of biochemical reactors must
preclude foreign
contamination (sterility); it should be able to operate under
aseptic
conditions for a specific time(days, if required).
(b)  Optimal mixing or agitation with low, uniform shear
(c) Adequate mass transfer (oxygen) or aeration for proper growth of
culture
(d) Power consumption should be as low as possible
(e) A system of temperature control should be provided
(f) A system of pH control should be provided
(g) Sampling facilities should be provided
(h) The vessel should be designed to require the minimal use of
labour in operation, harvesting, cleaning and maintenance

(i) Compliance with design requirements such as: ability to be sterilized; simple construction; simple measuring, control,
regulating techniques; scale up; flexibility; long term stability; compatibility with up- downstream processes; antifoaming
measures.
(j) Should follow containment regulation
-----GILSP—Good Industrial Large Scale Practices
-----Containment levels 1, 2, 3.
Fermentation technologies are of two types:
I. Liquid Fermentation
II. Solid State Fermentation

I. Liquid Fermentation is a controlled process which consists of growing cells in a liquid broth. In a
bioreactor, used for liquid fermentation, sterility of the material can be maintained and control of
temperature, pH, dissolved oxygen, and mixing, is possible. This will provide an optimal
environment for the specific microorganism to grow. Liquid fermentation systems are of two types:

1. Static culture --In static liquid fermentation

no mixing occurs, &
2. Submerged fermentation—In submerged
liquid fermentation, the liquid broth is
mixed.
II. Solid state fermentation is the aerobic growth of
microorganisms on solid substrates under limited water
conditions. The solid matrix, which provides the structure to
the material, may consist of organic-based materials (cereal
grains, wood chips, compost, cheese etc.) or inert materials
(perlite, vermiculite etc) impregnated with a liquid nutrient
broth.

Solid state bioreactor

 Fermentation
The word fermentation from a microbiological perspective is used to describe any biological process occurring under
anaerobic conditions (absence of oxygen). However, many researchers use the term to describe any process that uses a
microorganism to convert a particular medium into a specific product.

Two important aspects of microbial

growth which are of prime importance
in industrial microbiology are:-
1. The primary metabolites,
resulting from the log phase or
Trophophase; &

2. The secondary metabolites,

resulting from the stationary phase
or Idiophase.
 3 Types of Fermentation
Microbial fermentations in liquid media can be carried out under different operating conditions, i.e.,
batch growth, fed-batch growth or continuous growth.

Batch growth involves a closed system where all nutrients are present at the start of the fermentation
within a fixed volume. The only further additions may be acids or bases for pH control, or gases ( e.g.,
aeration, if required).

In fed-batch systems fresh medium or medium components are fed continuously, intermittently or are
added as a single supplement and the volume of the batch increases with time.

Continuous fermentations are open systems where fresh medium is continuously fed into the
fermentation vessel, but the volume remains constant as spent medium and cells are removed at the same
rate.
Batch fermentations
GROWTH PHASES AND SECONDARY METABOLITES
The growth of a bacterial culture can be represented by a curve that consists of four stages or phases:
Lag phase - growth and reproduction are just beginning
Log phase - reproduction is occurring at an exponential rate
Stationary phase - environmental surroundings and food supply cannot support any
more exponential growth
Death phase - when all of the nutrients have been exhausted, the population dies off
. Batch fermenters
Batch fermenters are used to carry out microbiological processes on a batch basis. There are a
number of steps involved. These are associated with the development of microorganisms from a
stock culture, and include agar slope & shake flask stages. Thereafter, this is followed by ‘seed’
& production stages.

Shake flask stage

Agar slope

Lab fermenter

Production stage
Advantages of Batch fermentations
1. Initial capital expenditure is less;
2. If contamination occurs, it is simple to terminate & restart a new batch.
Disadvantages of Batch fermentations
1. During batch fermentations certain environmental conditions continually change, particularly nutrient &
product concentrations , as well as specific growth rates. Hence steady states are not achieved.
2. Another disadvantage- Several stages are associated with operation of a batch fermenter:
 Charging of the fermenter with fresh medium;
 Sterilization of fermenter & medium;
 Inoculation of the fermentation;
 Production of microbial products;
 Harvesting of biomass & spent fermentation broth;
 Cleaning of the vessel.
And for all these, the fermentation vessel is not producing microbial products for a
considerable period of time. This non-productive period is referred to as the down-time
of the fermenter.
3. Batch-to-batch variability of the product.
4. Running costs are high.
5. More personnel required for operation.
Fed-batch fermentations
Fed- batch process is the enhancement of the closed batch process.
This is used in the production of substances such as penicillin & baker’s yeast.
In this process, substrate is added in increments as the fermentation progresses, which increases the
fermenter volume.
The formation of many secondary metabolites is subject to catabolite repression by high concentrations of
glucose, other carbohydrates, or nitrogen compounds. Hence, the critical elements of the nutrient solution are
added in small concentrations at the beginning of the fermentation & these substances continue to be added in
small doses during the production phase.
It is not possible to measure the substrate concentration directly & continuously during this operation,
hence indirect parameters (correlated with metabolism of critical substrate) are measured to control
feeding process.
For example:-For production of organic acids, the pH value is used to determine the rate of glucose feeding.
 Fed-batch culture requires special equipment such as a reservoir which holds the nutrients, pH modifiers so
that they can be added to the fermenter at regular intervals, and pumps to deliver culture medium aseptically to
the fermenter.
Fed batch mode is useful where a substrate causes viscosity problems or is toxic at high concentrations.
Fed-batch with recycle of cells(biomass) can also be used for some ethanol fermentations & waste-water
treatment processes.
 Advantages and disadvantages of the fed-batch reactors

1. The nutritional environment can be maintained approximately constant during the course
of the batch.
2. The production of by-products that are generally related to the presence of high
concentrations of substrate can also be avoided.
3. Since this method usually permits the extension of the operating time, high cell
concentrations can be achieved and thereby, improved productivity [mass of
product/(volume.time)].
4. Fed-batch might be the only option for fermentations dealing with toxic or low solubility
substrates.
Disadvantages:
it requires previous analysis of the microorganism, its requirements and the understanding
of its physiology with the productivity

it requires a substantial amount of operator skill for the set-up, definition and development
of the process
 Some examples of fed-batch use in industry

 Saccharomyces cerevisiae is industrially produced using the fed-batch technique so as to

maintain the glucose at very low concentrations, maximizing the biomass yield and
minimizing the production of ethanol, the chief by-product.
 The production of recombinant protein is achieved using fed-batch culture techniques. For
eg., Hepatitis B surface antigen (HbsAg) expressed in recombinant yeast.
Penicillin production is an example for the use of fed-batch in the production of a
secondary metabolite.
 The production of thiostrepton from Streptomyces laurentii .
The production of cellulase by Trichoderma reesei.
 Continuous fermentation

Culture vessel

A Simple Continuous Culture system

Continuous culture is an open system where fresh medium is continuously added &
culture is simultaneously removed at the same rate , resulting in a constant working
volume.

Cells grow exponentially for extended periods at a specified predetermined growth rate.

The system can reach a steady state in which the concentration of limiting nutrient & the
cell number do not vary with time.

 Rate of growth also depends on the Dilution Rate (D). D = F/ V; where F = Flow (L/hr),
V = reactor volume (L), D = dilution rate (per hr).

 Addition of fresh medium can be controlled at a fixed value, therefore the addition of the
rate-limiting nutrient is constant.

 This rate of medium input determines the growth rate & rate of loss of cells from
reactor (within certain limits).