APPLIED BIOTECHNOLOGYx

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Feb 20, 2013 (4 years and 5 months ago)

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APPLIED BIOTECHNOLOGY


Duration
-
3 Hrs



Max Marks: 70


Note:


1.

Answer
any EIGHT
questions from Section A.
Each
question carries
5
marks.

2. Answer any THREE questions from Section B.

Each question carries 10 marks.


Section A

Answer
any EIGHT
questions from Section A.
Each
question carries
5
marks.


1.

Define biotechnology and mention about 3 ancient biotechnology processes

Ans.

Biotechnology

(sometimes shortened to "
biotech
") is a field

of applied

biology

that involves the
use of living organisms and bioprocesses in

engineering
,

technology
,

medicine

and other fields requiring
bioproducts. Biotechnology also utilizes these products for manufacturing purpose. Modern

use of similar
terms includes

genetic engineering

as well as

cell

and

tissue culture
technologies. The concept
encompasses a wide range of procedures (and

history
) for modifying living organisms accor
ding to
human purposes


going back to domestication of animals, cultivation of plants, and "improvements" to
these through breeding programs that employ

artificial

selection

and

hybridization
. By comparison to
biotechnology,

bioengineering

is generally th
ought of as a related field with its emphasis more on higher
systems approaches (not necessarily altering or using biological materials

directly
) for interfacing with and
utilizing living things. The
United Nations

Convention on Biological Diversity

defines biotechnology as:
[1]

"Any technological application that uses biological systems, living organisms, or derivatives thereof, to
make or modify products or processes for specific use."

In other
terms: "Application of scientific and technical advances in life science to develop commercial
products" is biotechnology. Biotechnology draws on the pure biological sciences
(
genetics
,

microbiology
,

animal cell culture
,

molecular biology
,

biochemistry
,
embryology
,

cell biology
) and
in many instances is also dependent on knowledge and methods from outside the sphere of biology
(
chemical engineering
,

bioprocess engineering
,

information technology
,

biorobotics
). Conversely, modern
biological sciences (including even concepts such as

molecular ecology
) are intimately entwined and
dependent on the methods developed through biotechnology and what is commonly thought of as the

life
sciences

industry.


2.

Write a brief note on CPCSEA guidelines

3.

Write about equilibrium density
-
gradient centrifugation

Ans.

centrifugation
, also known as

density gradient centrifugation

or

equilibrium sedimentation

is a
technique used to separate

molecules on the basis of buoyant density. (The word "isopycnic" means
"equal density".) Typically, a "self
-
generating" density gradient is established via equilibrium
sedimentation, and then analyte molecules concentrate as bands where the molecule densi
ty matches
that of the surrounding solution. To illustrate the process, consider the fractionation of nucleic acids such
as

DNA
. To begin the analysis, a mixture of
caesium chloride

and DNA is placed in a

centrifuge

for several
hours at high speed to generate a force of about 10^5 x

g

(earth's gravity). Caesium chloride is used
because at a concentration of 1.6 to 1.8 g/mL it is similar to the density of DNA. After some time a
gradient of the caesium ions is formed, caused by two oppo
sing forces:

diffusion

and centrifugal force.
The sedimenting particles (caesium

ions) will sediment away from the rotor, and become more
concentrated near the bottom of the tube. The diffusive force arises due to the concentration gradient of
solvated caesium chloride and is always directed towards the center of the rotor. The balanc
e between
these two forces generates a stable density gradient in the caesium chloride solution, which is more
dense near the bottom of the tube, and less dense near the top.

The DNA molecules will then be separated based on the relative proportions of AT
(
adenine

and

thymine

base pairs) to GC (
guanine

and
cytosine

base pairs). An AT base pair has a lower
molecular weight than a GC base pair and therefore, for two DNA molecules of equal length, the one with
the greater proportion of AT base pairs w
ill have a lower density, all other factors being equal. Different
types of nucleic acids will also be separated into bands, e.g. RNA is denser
than

supercoiled

plasmid

DNA, which is denser than linear chromosomal DNA.

4.

How do you increase the monoclonal antibody titre in a culture


5.

Distinguish between mutualism and Parasitism?

Ans.

Mutualism

is the way two organisms

of different species

biologically interact

in a relationship in
which each individual derives a

fitness

benefit (
i.e.
, increased or improved reproductive output). Similar
interactions within a species are known as

co
-
operation
. M
utualism can be contrasted with
interspecific
competition
, in which each species experiences

reduced

fitness, and

exploitation
, or
parasitism
, in which
one species benefits at the

expense

of the other. Mutualism is a type of
symbiosis
. Symbiosis is a broad
category, defined to include relationships that are mutualistic,
parasitic
, or

commensal
. Mutualism is only
one

type
.

A well known example of mutualism is the relationship between

ungulates

(such as

cows
)
and
bacteria

within their

intestines
. The ungulates ben
efit from the

cellulase

produced by the bacteria,
which facilitates

digestion
; the bacteria benefit from having a
stable supply of

nutrients

in
the

host
environment.

Parasitism

is a type of non mutual relationship between

organisms

of
different

species

where one organism, the

parasite
, benefits at the expense of the other, the

host
.
Traditionally

parasite
referred to organisms with lifestages that needed more than one host (e.g.

Taenia
solium
). These are now called

macroparasites

(typically

protozoa

and

helminths
). The word

parasite

now
also refers to

microparasites
, which are typically smaller, such as

viruses

and

bacteria
, and can be directly
transmitted between hosts of the same species

[1]
. Examples of parasites include the plants mistletoe and
cuscuta, and organisms such as leeches.

Unlike

predators
, parasi
tes are generally much smaller than their host; both are special cases
of
consumer
-
resource interactions
.
[2]

Parasites show a high degree of

specialization
, and

reproduce
at a
faster rate than their hosts. Classic examples of parasitism include interactions between
verte
brate

hosts
and diverse animals such as

tapeworms
,

flukes
, the

Plasmodium

species, and
fleas
.

Parasitism is differentiated from the

parasitoid

relationship,
though not sharply, by the fact that parasitoids
generally kill or sterilise their hosts. Parasitoidism occurs in much the same variety of organisms that
parasitism does.

The harm and benefit in parasitic interactions concern the

biological fitness

of the organisms involved.
Parasites reduce host fitness in many ways, ranging from general or specialized

pathology
, such
as

parasitic castration

and impairment of

secondary sex characteristics
, to the modification of host
behaviour. Parasites increase their fitness by exploiting hosts for resources necessary for

the parasite's
survival, e.g. food, water, heat, habitat, and genetic dispersion.



6.

What is bioremediation?

Ans.

Bioremediation

is the use of

microorganism

metabolism to remove
pollutants. Technologies can be
generally classified as

in situ

or

ex situ
.
In situ

bioremediation involves treating the cont
aminated material
at the site, while

ex situ

involves the removal of the contaminated material to be treated elsewhere. Some
examples of bioremediation technologies
are

phy
toremediation
,

bioventing
,

bioleaching
,

landfarming
,

bioreactor
,
composting
,

bioaugmentation
,

rhizo
filtration
, and

biostimulation
.

Bioremediation can occur on its own (natural attenuation or intrinsic bioremediation) or can be spurred on
via the addition of fertilizers to increase the bioavailability within the medium (biostimulation). Recent
ad
vancements have also proven successful via the addition of matched microbe strains to the medium to
enhance the resident microbe population's ability to break down contaminants. Microorganisms used to
perform the function of bioremediation are known as

bio
remediators
.
[1]

Not all contaminants, however, are easily treated by bioremediation using microorganisms. For
example,

heavy metals

such as

cadmium

and
lead

are not readily absorbed or captured by
microorganisms. The assimilation of m
etals such as

mercury

into the

food chain

may worsen
matters.

Phytoremediation

is useful in these circumstances because natural plants or

transgenic
p
lants

are able to

bioaccumulate

these toxins in their above
-
ground parts, which are then harvested for
removal.
[2]

The heavy metals in the harvested biomass may be further concentrated by incineration or
even recycled for industrial use.

The elimination of a wide range of pollutants and wastes from the environment requires increasing our
understanding of
the relative importance of different pathways and regulatory networks to

carbon flux

in
particular environments and for particular compounds, and they will certainly accelerate the d
evelopment
of bioremediation technologies and

biotransformation

processes.


7.

How are PCBs biodegraded?

Ans.

A

printed circuit board
, or

PCB
, is used to mechanically support and electrically connect
electronic
components

using

conductive

pathways, tracks or signal traces

etched

from
copper

sheets

laminated

onto
a non
-
conductive

substrate
. It is also referred to as

printed wiring board

(
PWB
) or

etched
wiring board
.
A PCB populated with electronic components is a

printed circuit assembly

(
PCA
), also known as
a

printed circuit board assembly

or

PCB Assembly

(
PCBA
). Printed circuit boards are used in virtually
all but the simplest commercially produced ele
ctronic devices.

Alternatives to PCBs include

wire wrap

and

point
-
to
-
point const
ruction
. PCBs are often less expensive
and more reliable than these alternatives, though they require more layout effort and higher initial cost.
PCBs are much cheaper and faster for high
-
volume production since production and soldering of PCBs
can be done

by automated equipment. Much of the electronics industry's PCB design, assembly, and
quality control needs are set by standards that are published by the

IPC

organizatio
n.

Hanson, described flat foil conductors laminated to an insulating board, in multiple layers.

Thomas
Edison

experimented with chemical methods of plating conductors onto linen paper in 1904. Arthur Berry
in 1913 patented a print
-
and
-
etch method in Britain, and in the United States Max Schoop obtained a
patent
[1]

to flame
-
spray metal onto a board through a patterned mask. Charles Durcase in 1927 patented
a method of electroplating circuit patterns.

[2]

The Austrian Jewish engineer

Paul Eisler

invented the printed circuit while working in England around
1936 as part of a

radio

set. Around 1943 the USA began to use the technology on a large scale to
make

proximity fuses

for use in

World War II

[2]
. After the war, in 1948, the USA released the invention for
commercial use. Printed circuits did not become commonplace in

consumer electronics until the mid
-
1950s, after the

Auto
-
Sembly

process was developed by the

United States Army
.

Before printed circuits (and for a while after their
invention),

point
-
to
-
point construction

was used. For
prototypes, or small production runs,
wire wrap

or

turret board

can be more efficient. Predating the printed
circuit invention, and similar in spirit, was

John Sargrove
's 1936

1947 Electronic Circuit Making
Equipment (ECME) which sprayed metal onto a

Bakelite

plastic board. The ECME could produce 3 radios
per minute
.


8.

What are the characteristics of wastewater?

Ans.

Wastewater

is any

water

that has been adversely affected in quality by

anthropogenic

influence. It
comprises liquid waste discharged by domestic residences, commercial properties, industry, and/or
agriculture and ca
n encompass a wide range of potential contaminants and concentrations. In the most
common usage, it refers to the municipal wastewater that contains a broad spectrum of contaminants
resulting from the mixing of wastewaters from different sources.

Sewage

is

correctly the subset of wastewater that is contaminated with

feces

or

urine
, but is often used to
mean any waste water. "
Sewage
" includes domestic, municipal, or industrial

liquid waste
products

disposed of, usually via a

pipe

or

sewer

or similar structure, sometimes in a
cesspool emptier
.

The physical infrastructure, including pipes,

pumps
, screens, channels etc. used to convey
sewage from
its origin to the point of eventual treatment or disposal is termed

sewerage
.

Wastewater or sewage can come from (text in brackets indicates likely inclusions or contaminants):



Human waste

(
fæces
, used

t
oilet paper

or wipes,

urine
, or other bodily fluids), also known
as
blackwater
, usually from

lavatories
;



Cesspit

leakage;



Septic tank

discharge
;



Sewage treatment

plant discharge;



Washing water (personal, clothes, floors, dishes, etc.), also known as

greywater

or

sullage
;



Rainfall collected on roofs, yards, hard
-
standings, etc. (generally clean with traces of

oils

and
fuel
);



Groundwater

infiltrated into sewage;



Surplus manufactured liquids from domestic sources (drinks, cooking oil,

pesticides
,
lubricating
oil
,

paint
, cleaning liquids, etc.);



Urban

rainfall

runoff from

roads
, carparks, roofs, sidewalks, or pavements (contains oils, animal
fæces,

litter
, fuel or

rubber

residues,

metals

from vehicle

exhausts
, etc.);


9.

Write about the physical properties and chemical composition of culture media

10.

Discuss polymerase chain reaction?

Ans.

The

polymerase chain reaction

(
PCR
) is a

scientific technique

in

molecular biology

to

amplify

a
single or a few copies of a piece of

DNA

across several orders of magnitude, generating thousands to
millions of copies of a particular

DNA sequence
.

Developed in 1983 by

Kary Mullis
,
[1]

PCR is now a common and often indispensable technique used in
medical and biological research labs for a variety of applications.
[2]
[3]

These include

DNA
cloning

for

sequencing
, DNA
-
based

phylogeny
, or functional analysis of

genes
; the diagnosis of

hereditary
diseases
; the identification of

genetic fingerprints

(used in

forensic sciences

and

paternity testing
); and the
detection and diagnosis of

infectious diseases
. In 1993, Mullis was awarded the

Nobel Prize in
Chemistry

along with

Michael Smith

for his work on
PCR.
[4]

The method relies on

thermal cycling
, consisting of
cycles of repeated heating and cooling of the reaction
for

DNA melting

and

enzymatic

replication

of the DNA.
Primers

(short DNA fragments) containing
sequence
s complementary to the target region along with a

DNA polymerase

(after which the method is
named) are key components to enable selective and repeated amplification. As PCR pro
gresses, the
DNA generated is itself used as a template for replication, setting in motion a

chain reaction

in which the
DNA template is

exponentially

amplified. PCR can be extensively modified to perform a wide array
of

genetic manipulations
.

Almost all

PCR applications employ a heat
-
stable DNA polymerase, such as

Taq polymerase
, an enzyme
originally isolated from the bacterium

Thermus aquaticus
. This DNA polymerase

enzymatically

assembles
a new DNA strand from DNA building
-
blocks, the
nucleotides
, by using single
-
stranded DNA as a template
and

DNA oligonucleotides

(also called

DNA primers
), which are required for initiation of DNA synthesis.
The vast majority of PCR methods use

thermal cycling
, i.e., alternately heating and cooling the PCR
sample to a defined series of temperature steps. These thermal cycling steps are necessary first to
physically separate the two strands in a DNA double helix at a high temperat
ure in a process called

DNA
melting
. At a lower temperature, each strand is then used as the

template

in DN
A synthesis by the DNA
polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use
of

primers

that are
complementary

to the DNA region targeted for amplification under specific thermal
cycling conditions.


Answer any THREE questions from Section B. Each question carries 10 marks.

Section
B


11.

Debate reproductive cloning in animals versus humans

Ans.

Human

animal communication is easily observed in everyday life. The interactions between pets
and their owners, for example, reflect a form of spoken, while not necessarily verbal dialogue. A dog

being scolded does not need to understand every word of its admonishment, but is able to grasp the
message by interpreting cues such as the owner's stance, tone of voice, and

bo
dy language
. This
communication is two
-
way, as owners can learn to discern the subtle differences between barks and
meows … one hardly has to be a professional animal trainer to tell the difference between the bark of an
angry dog defending its home and th
e happy bark of the same animal while playing. Communication
(often nonverbal) is also significant in

equestrian

activities such as

dressage
.

[
edit
]
Word repetition in birds

Although the

word repetition

skills observed in

some birds

(most famously

parrots
) should not be mistaken
for lingual communication, this tendency has nonetheless influenced fictional portrayals of animal
communication, as sentient talking parrots and similar birds are common in children's fiction, such
as the
talking, loud
-
mouth parrot

Iago

of Disney's

Aladdin
.

Bruce Thomas Boehner
's book

Parrot Culture: Our
2,500
-
Year
-
Long Fascination with the World's Most Talkative B
ird

explores this issue thoroughly.

[
edit
]
The next level: language

Achieving a deeper level of
communication between animals and humans has long been a goal of
science. Perhaps the most famous example of recent decades has been

Koko
, a gorilla who is
supposedly able to c
ommunicate with humans using a system based on
American Sign Language

with a
"vocabulary" of over 1000 words.

[
edit
]
John Lilly and cetacean communication

In the 1960s,

John Lilly
, M.D., prolific writer and explorer of

consciousness

via the

isolation tank

(his
invention), and contemporary and associate of

Timothy Leary
, began experiments i
n the

Virgin
Islands

aiming to establish meaningful communication between humans and the
bottlenose
dolphin

(
Tursiops truncatus
). Lilly financed, mostly personally, a human
-
dolphin cohabitat, a house on the
ocean's shore that contained an area that was part
ially flooded and allowed a human and dolphin to live
together in the same space, sharing meals, play, language lessons, and even sleep.

Two experiments of this sort are explained in detail in Lilly's popular books (see

John Lilly

for
bibliography). The first experiment was more of a test run to check psychological and other strains on the
human and cetacean participants, determining the extent of the need for other human contact,

dry
clothing, time alone, and so on. Despite tensions after several weeks, the experimenter, Margaret C.
Howe, agreed to a two
-
and
-
a
-
half month experiment, living isolated with 'Peter' dolphin.


12.

List the gene delivery methods with special mention to
microinjection

Ans.

Microinjection

refers to the process of using a glass

micropipette

to insert substances at
a

microscopic

or borderline

macroscopic

level into a single living

cell
. It is a simple mechanica
l process in
which a needle roughly 0.5 to 5 micrometers in diameter penetrates the

cell membrane

and/or the

nuclear
envelope
. The desired contents are then injected into the desired sub
-
cellular compartment and the
needle is removed. Microinjection is normally performed under a specialized

optical microscope

setup
called a

micromanipulator
. The process is frequently used as
a

vector

in

genetic
engineering

and

transgenics

to insert

genetic material

into a single cell. Microinjection can also be used in
the

cloning

of organisms, and in the study of cell biology and viruses. Microcapillary and microscopic
devices are used to deliver DNA into a protoplast.

[
edit
]
Examples



Microinjection is used as a vector in

transgenic plant production
.



Microinjection of genes into fertilized

eggs

is a common vector used in the production of higher forms
of

transgenic animals
.



Microinjection of a

gene knockdown

reagent such as a

morpholino

oligo into

eggs

or early

zygotes

is
commonly used to probe the function of a

gene

during development of

embryos
.


13.

Define the usage of progesterone, prostoglandin and PMSG

Mention the importance of marker genes in DNA transfection


Ans.

Transfection

is the process of deliberately introducing nucleic acids into cells. The term is used
notably for non
-
viral methods in eukaryotic cells
[1]
. It may also refer to other methods and cell types,
although other terms are preferred: "
transformation
" is more often used to describe non
-
viral

DNA

transfer
in

bacteria
, non
-
animal

eukaryotic

cells and plant cells
-

a distinctive sense of transformation refers to
spontaneous genetic modifications (
mutations

to cancerous

cells (
carcinogenesis
), or under stress (UV
irradiation)).

Transduction

is
often used to describe virus
-
mediated DNA transfer. The word

transfection

is
a

blend

of

trans
-

and

infection
.

Genetic

material

(such as

supercoiled plasmid DNA

or

siRNA

constructs), or even

proteins

such
as

antibodies
, may be transfected.

Transfection of

animal cells

typically involves

opening transient pores or "holes" in the

cell membrane
, to
allow the uptake of material. Transfection can be carried out using

calcium phosphate
, by

electroporation
,
or by mixing a

cationic

lipid

with the material to produce
liposomes
, which fuse with the cell membrane and
deposit their cargo inside.

Transfection ca
n result in unexpected morphologies and abnormalities in target cells.

The meaning of the term has evolved.
[2]

The original meaning of transfection

was "infection
by transformation," i.e., introduction of DNA (or RNA) from a

prokaryote
-
infecting virus
or

b
acteriophage

into cells, resulting in an infection. Because the term transformation had
another sense in animal cell biology (a genetic change allowing long
-
term propagation in
culture, or acquisition of properties typical of cancer cells), the term tra
nsfection acquired, for
animal cells, its present meaning of a change in cell properties caused by introduction of
DNA.

14.

Why cell Ca2+ is an inexpensive but cAMP is an expensive second messenger?