Assignment 3

IMMUNOLOGY AND BIOTECHNOLOGY

Sub.To : Dr. Nidhi Srivastva

By: Piyush Sharma

Unit III

Q.1: Biotechnology: it is a branch of science which deals with the application of biological animals along with modern
technology to obtain commercial benefit and enhance products of human benefits.

Scope of Biotechnology:

a. Screening of therapeutic agents.

b. Bio-processing of alkenes inro useful glycols and oxides.
c. Development of immobilized cells and enzymes for chemical process industries.
d. Engineering of organisms for specific purposes.
e. Improvement of micro-organisms for obtainting quality products.
f. Human gene therapy.
g. Production of vitamins.
h. Ethanol manufacturing.
i. Production of human insulin and interferons.
j. Production and development of vaccines.
k. Use of bio-agents (bio-pesticides etc.).
l. Making diagnostic kits for detecting infection from pathoens.
m. Production of transgenetic plants and animals.
n. Production of monoclonal Ab.

Application of biotechnology: Applications of biotech is divided and termed as red, green & grey/white biotechnology
according to their area

Red Biotechnology: it refers to the application of biotechnology in the field of human & animal medicine and
pharmacology. Biotechnology is used in medical as

1. For biopharmaceutical drug development: development of human proteins for therapeutic purposes using
recombination technology. For eg. Recombinant human insulin.
2. Development of drug delivery agent: various drugs fail to reach their target sites due to less stability. For
drugs like that an efficient drug delivery system is developed.

Green Biotechnology: it refers to the application of biotechnology in the field of agriculture

1. Transgenic plants: plants can be genetically modified to produce a variety with high yield and quality food
products.
2. Transgenic animals: these animals are recombined with genes of interest to increase yield and quality of
their respective products.
3. Livestock breeding: the transgenic organisms then are breeded to obtain more efficient variety:

Grey/white biotechnology: it refers to the application of biotechnology for industrial or environmental purpose.

1. Technical enzymes: technical enzymes include proteases, lipases etc which are present in detergents to help
dissolving fats and proteins.
2. Fungus Aureobasidium pullulans degrades polyurethane.
Q.2: Animal cell culture: cell culture refers to process by which the cells are grown outside a body in necessary
conditions. In cell culture cells are removed from an animal and are grown in vitro I n favorable conditions. Types of
animal cell culture:

It can be divided on the basis of different criteria:

A. On the basis of source from which cell is obtained:

a. Primary cell culture: this refers to the cell cultured from cells directly obtained from the host tissue.
Cells are first dissociated from parent tissue and then grown on a suitable medium cell culture thus
obtained is called primary cell culture. These culture usually consists of heterogenous mixture of cells
which divide for a limited time but are similar to their parent. Primary cell culture grows by two types:
i. Adherent cell culture: these cells are contact dependent and grow as a monolayer. The cells
require to remain attached to a solid or semi-solid substrate for proliferation. Epithelial cells and
fibroblast grows as adherent cell culture. If the bottom of culture dish is completely covered by
a layer of cell usually one cell thick, the cells stop proliferating. Thses cells can be easily
transferred to other dishes for sub culture. Replacement of medium is required. Lag phase of
cell growth is long.
ii. Suspension cell culture: in this type the cells do not grow attached to the vessel surface so
rthese are anchorage independent or non-adherant cells. These cells grow floating in the
medium. These cells grow faster and are easily maintained. Replacement of medium in not
required. Lag period of cell growth is short.

Confluence and sub-culturing: confluence refers to the state when cultured cells use all the
substrate available for growing and dish becomes crowded which may lead to cell death. In such
cases sub-cultures of these cells are grown where cells from primary cultures are transferred to
new culture dishes.

b. Secondary cell culture: subcultured primary culture is called secondary culture. This process includes
removing culture media and disassociating adhered cells enzymatically.
On the basis of span of culture cell culture are of 2 types
a. Finite cell line: the cells which undergo a limited no. of cell division and has a limited life span are
known as finite cell lines. These cells proliferate for limited time and then loses the ability to
proliferate stage called senescence. Mostly cells are finite cell lines.
b. Continuous cell line: if a finite cell line transforms and acquire the ability to divide indefinitely it
becomes continuous cell line. These cells are less adherent, fast growing and grow up to higher
density. Malignant and cancerous cells give rise to continuous cell line.

Q.3. Recombinant DNA technology: it is also called gene cloning or molecular cloning it refers to a general procedure of
transfer of genetic material from one organism to another. a recombinant DNA experiment often has the following
format:
1. The DNA (cloned DNA, insert DNA, target DNA, or foreign DNA) from a donor organism is extracted,
enzymatically cleaved (cut, or digested), and joined (ligated) to another DNA entity (a cloning vector) to form a new,
recombined DNA molecule (cloning vector– insert DNA construct, or DNA construct).
2. This cloning vector–insert DNA construct is transferred into and maintained within a host cell. The
introduction of DNA into a bacterial host cell is called transformation.
3. Those host cells that take up the DNA construct (transformed cells) are identified and selected (separated, or
isolated) from those that do not.
4. A DNA construct can be created so that the protein product encoded by the cloned DNA sequence is
produced in the host cell.
Materials required:

1. Source DNA: the source DNA is obtained from a target organism from which the gene of interest is to be
obtained. The gene is sliced of the DNA with the help of restriction enzymes.
 2. Cloning vector: it is a fragment of DNA that can be maintained in an organism and into which another DNA
fragment can be inserted and can be cloned. Cloning vectors can be obtained from bacteria, virus yeast etc.

Features of a cloning vector: Origin of replication> a specific sequence of nucleotides in a DNA molecule
from where replication initiates.

Selectable Marker> vector must poses a selectable marker gene which aloow selection of succesfylly
transformed cells.

Restriction sites> vector must poses restriction sites on which restriction enzymes can cleave the DNA.

It must not be too big.

Stable> its replication properties must not be impaired when foreign DNA is inserted.

Types: vectors are of following types>

a. Plasmids: Plasmids are self-replicating, double-stranded, circular DNA molecules that are maintained in
bacteria as independent extra-chromosomal entities. The size ranges from 1kb-500kb. Dna fragment that
can be cloned using plasmid ranges from 0.1-10kb. Selectable marker is antibiotic resistance. (pBR322).
b. Bacteriophage λ vector: bacteriophage vector is obtained from bacteriophage lambda which infects E.coli. it
is used for cloning DNA fragment ranging from 15-20 kb. Bacteriophage DNA has 2 cos (cohesive) sites at
each end.
c. Cosmid: cosmids are used for cloning large DNA segments of size 30-50kb. It combines the feayure of both
plasmid and lambda phage.
d. BAC (Bacterial artificial Chromosome): Cloning limit is 30-300kb. Marker is chloramphenicol and lactose
mobilizing gene. It is a modification of f-plasmid and is synthesized artificially. (pUvBBAC)
e. YAC (Yeast artificial chromosome): cloning limit is 100-1000kb. It is artificial vector consisting of yeast
centromere. It is isolated from S. cerevisiae.
f. Human Artificial chromosome: it is used to clone human genes and can carry large segment of DNA.

DNA ligase enzyme is required to join cleaved DNA with vector.

Procedure:

1. Isolation of the target gene: target gene or gene of interest is first cleaved using restriction enzymes and
then purified using different methods. Gene can also be isolated using reverse transcriptase enzyme.
2. Vector selection: vectors are selected according to the requirement of cloning size. The vectors are then
cleaved by same restriction enzyme.
3. Joining the target gene with vector: target gene and vector is mixed and joined using DNA ligase enzyme
and thus a DNA recombinant is made.
4. Transforming the recombinant vector to host cell: recombinant vector thus formed is then transferred into
host cell.
5. Isolation of recombinant cell: the host cell is then grown in the culture dish and the successfully
transformed cells are selected using the marker gene.
Q.4: Construction of DNA libraries: (gene bank, clone bank) it refers to a collection of DNA fragments that is
stored and propagated through the process of molecular cloning. There are two types of DNA libraries:

1. Genomic DNA library: it refers to the DNA collection library of whole genome of the organism. The genome is
first subdivided into many fragments and then is inserted into host bacterial cells by rDNA technology.

Construction:

a. To create a Genomic DNA library DNA from source organism is first isolated and purified.
b. The DNA is then cleaved into small fragments by using restriction enzymes.
c. The target DNA fragments are then ligated to cloning vectors.
d. Vectors are then transferred into the host cell for cloning.
e. The bacterial cells consisting of target DNA are then identified and maintained.
f. Host cell DNA identification is done by different methods
i. DNA hybridization with a labeled DNA probe
ii. immunological screening for the protein product
iii. assaying for protein activity
iv. functional (genetic) complementation
3. cDNA library: cDNA stands for complimentary DNA and it is DNA library of only the active genes of an
organism, organ, tissue or cell. In cDNA library only the coding or genes those are transcribed are
maintained in the host cell. cDNA is double stranded DNA constructed from functional mRNA by the process
of reverse transcription.
Construction:
a. mRNA from the host organism is isolated and purified. Processed mRNA consists of a poly A tail which
helps in the purification. Cellulose beads are conjugated with poly thymidine residue which binds to
poly A tail of mRNA purifying the latter.
b. Before cloning into avector mRNA needs to be coverted into ds DNA.
c. Reverse transcriptase enzyme helps in synthesizing the first DNA strand from the mRNA.
d. DNA polymerase-1 from E. coli helps in synthesizing the 2nd DNA strand.