CHAPTER 11

BIOTECHNOLOGY: PRINCIPLES AND

PROCESSES
1. What is Genetic engineering?

Ans. Genetic engineering is the technique mainly used to change or to

modify the genetic material (DNA/RNA), and to introduce them into
other organisms. It is the process of artificial synthesis, isolation,
modification, combination, addition and repair of genetic material as
needed.

2. What are the two core techniques that enabled the birth of
modern biotechnology?
Ans. The two core techniques that enabled the birth of modern
biotechnology are:
1)Genetic engineering
2)Bioprocess engineering

Genetic engineering
The modification of the genetic material (RNA or DNA) of organisms to
use them to produce commercially important substances is called
genetic engineering.

Bioprocess engineering
Maintenance of sterile ambience in chemical engineering processes to
enable growth of only the desired microbe or eukaryotic cell in large
quantities for the manufacture of biotechnological products like
antibiotics, vaccines, enzymes, etc., is called bioprocess engineering.

3. Mention two classes of restriction enzymes. Suggest their

respective roles.
Ans. Exonucleases and endonucleases. Exonucleases remove nucleotides
from the ends of the DNA. Endonucleases cut DNA at specific sites
between the ends of DNA.

4. Define the following:

(a) Palindrome
(b) Sticky ends
(c) Recognition sequence
(a) The palindrome in DNA is a sequence of base pairs that reads
same on the two strands when orientation of reading is kept the
same.
e.g.
5' —— GAATTC —— 3'
3' —— CTTAAG —— 5'

(b)When restriction enzymes cut the strand of DNA a little away from
the centre of the palindrome sites, but between the same two
bases on the opposite strands, overhanging stretches (single
stranded portions) are generated. These are called sticky ends.
(c) Recognition sequences are the 4 to 8 nucleotide long sequences
that restriction enzymes identify and are distinguished by a specific
form of internal symmetry. The recognition sequence site is
recognized by restriction endonucleases and they cleave the DNA
at or near the recognition site.

5. What are three basic steps in genetically modifying an organism?

Ans. The three basic steps in genetically modifying an organism are as
follows:
(i) Identification of DNA with desirable genes;
(ii) Introduction of the identified DNA into the host;
(iii) Maintenance of introduced DNA in the host and transfer of the DNA
to its progeny.
6. Give the diagrammatic representation showing the steps in
formation of recombinant DNA by action of restriction
endonuclease enzyme – EcoRI.
Ans.

7. What are the properties of a good vector?

Ans. A good vector must possess the following properties:

1. The vector must be small in size so that it is easy to isolate and

purify.
2. It should have an origin of replication (ori), a base pair sequence
where replication starts.
3. It should have a selectable marker that helps in selecting the
transformed host cells.
4. The vector should have at least one unique recognition site to bind
the foreign DNA.
8. (a) Draw a neat and labelled diagram of vector pBR322. Which
are the two selectable markers present in it.
(b) Identify the significance of Origin of replication.

Ans. (a)

The two selectable markers present in it are amp R and tetR.

(b) The ori is the place where DNA replication begins, enabling a plasmid
to reproduce itself. It determines the vector copy number and is
responsible for controlling the copy number of the linked DNA.

9. What are ‘Selectable markers’? What is their use in genetic

engineering?

Ans. A selectable marker is a gene which helps in selecting those host

cells which contains the vector, eliminating the non–transformants e.g. –
gene encoding resistance to antibiotics are useful selectable markers as
they allow selective growth of transformants only.
10. How are recombinants selected?
Ans. A marker gene helps in differentiating between transformants and non-
transformants. This helps in selecting the suitable recombinants as well. In
case of E-coil; pBR322 is the vector which contains two selectable markers;
one conferring resistance to antibiotic ampicillin and one conferring
resistance to antibiotic tetracycline. The ligation of alien DNA is carried out
at a restriction site (e.g. BamHI) present in tetracyline resistance gene.
The presence of ampicillin gene in pBR322 will result in resistance to
ampicillin. Therefore the transformants can be selected by growing on a
medium containing ampicillin.
The insertional inactivation of tetracycline gene in pBR322 will result in loss of
resistance to tetracycline by E.coli. This can be found out by growing the
recombinants on two plates; one containing tetracycline and another
containing ampicillin. The recombinants will grow in ampicillin but not in
tetracycline.

11. For selection of recombinants, insertional inactivation of antibiotic

resistance is a marker that has been superceded by insertional
inactivation of a marker gene coding for a chromogenic substrate.
Give reasons.

Ans. Selection of recombinants using insertional inactivation of antibiotic

markers is a tedious process. This is because it requires simultaneous
plating of transformants on two different antibiotic containing mediums.

A marker gene for chromogenic substrate helps in identifying the

recombinant DNA on the basis of gain or loss of colour from the
chromogenic substrate. Selection of recombinants using insertional
inactivation of a marker gene coding for a chromogenic substance is an
easy method as it requires plating only on one medium. Insertional
inactivation of such marker gene will result in no blue colour imparted in the
colony. Thus, the recombinants will produce white colonies while the
non-recombinants will produce blue colored colonies in the presence of
chromogenic substrate which makes it easy to identify recombinants and
non-recombinants by visual screening.
12. Name and describe the technique that helps in separation and
isolation of DNA fragments.
Ans. Gel electrophoresis: It is a technique of separation of charged
molecules, under the influence of an electrical field, so that they migrate
in the direction of electrode bearing the opposite charge, through a
medium/matrix.
After the cutting of DNA by restriction enzymes, fragments of DNA are
formed. These fragments are separated by gel electrophoresis. The most
commonly used matrix is agarose, which is a polysaccharide extracted
from sea weeds. DNA fragments separate according to size, through the
pores of agarose gel.
The separated DNA fragments can be seen only after staining DNA with
a compound ethidium bromide (EtBr), as bright orange colored bands,
on exposure to UV radiations. The separated bands of DNA are cut out
from the agarose gel and extracted from the gel piece. This step is called
as elution.

13. How is amplification of the gene of interest carried out?

Ans. Polymerase chain reaction (PCR) is the process by which the
amplification of the gene of interest is carried out with two sets of primers
and a thermostable DNA polymerase enzyme, Taq polymerase.

The process involves the following steps:

Denaturation: The double-stranded DNA is heated up to 94oC which causes
the hydrogen bonds to break and the two strands get separated.
Annealing: The two sets of primers are added which bind to the appropriate
complementary segment of the DNA strand at 54oC.
Extension: The Taq polymerase enzyme polymerizes the nucleotide chain
using the nucleotides provided in the medium and by using the template
strand at 72oC.

14. Describe the various steps involved in Recombinant DNA

technology.

Ans. The process of recombination DNA technology consists of the following

steps:

1. Isolation of genetic material (DNA)

DNA is enclosed within the membrane. So, to extract it out, the cell wall is
broken down to release the genetic material. This can be done by using cell
wall digesting enzymes such as lysozymes for bacterial cells, cellulose for plant
cells and chitinase for fungal cells.

2. Cutting of DNA at specific locations

This is done by using restriction enzymes that cut DNA at specific recognition
sequences and give the desired DNA fragments.

3. Isolation of desired DNA fragment: The gene of interest is isolated

by gel electrophoresis.
4. Amplification of gene of interest: Multiple copies of the gene of
interest is synthesised in vitro using PCR.
5. Joining of DNA fragment: The gene of interest and the vector are
joined together with the help of the enzyme DNA ligase. The
cloning vector is cut down by the same restriction enzyme used for
cutting the foreign DNA so that they both generate similar sticky ends.
This is necessary for efficient ligation.
 6. Insertion of DNA into the host cell: The recombinant DNA can be
inserted into the host cell by transformation, Recipient cells are made
‘competent’ to receive, take up DNA present in its surrounding.
Recombinant DNA is introduced into a suitable host cell by vector
– based or vector – less method.
7. Selection and screening of transformed cells: The selection and
screening of transformed cells can be by any of the following methods-
blue-white screening or insertional inactivation using antibiotic
resistance gene as selectable marker.
8. Obtaining the foreign Gene product: After having cloned the
gene of interest and having optimized the conditions to induce
expression of the target protein, one has to consider producing it
on large scale. For this bio reactors are used. The cultures are then
used for extracting the desired protein. These proteins (called as
recombinant proteins) are purified by using different separation
techniques.
15. What is “Insertional Inactivation”?
Ans. If a recombinant DNA is inserted within the coding sequence of
enzyme –galactosidase, then it results into inactivation of that gene
thereby producing no enzyme. This is referred to as “Insertional
Inactivation”. The presence of chromogenic substrate gives blue–
coloured colonies if the plasmid in bacteria does not have an insert.
Presence of insert (Gene of interest) results into insertional inactivation
and the colonies do not produce any colour.
16. Why is making cells competent essential for biotechnology
experiments? How bacterial cells are made competent?

Ans. Competency is the ability of a cell to take up foreign DNA.

Bacteria or any cell (animal or plant) cannot take up foreign DNA

naturally from the surroundings. This is because DNA is
a hydrophilic molecule and cannot pass through the membrane which is
hydrophobic in nature. For recombinant DNA integrated into a vector, it
is necessary for the DNA to be introduced in the cell. Therefore, making
the host cells competent is necessary in biotechnology experiments.

Cells can be made competent by treatment with divalent cations like

calcium followed by heat shock as follows:

Chemical action: The bacterial cell is treated with a specific

concentration of divalent cation, i.e. calcium. It increases the attachment
of foreign DNA with the cell.

Heat shock treatment: The bacterial cells are incubated with

recombinant DNA on ice, followed by brief treatment of heat at 42°C and
again putting them back on ice. This increases the efficiency of DNA to
enter it through pores in cell membrane.

17. List two methods by which recombinant DNA is introduced in

animal and plant cells respectively.

Ans. Two methods by which recombinant DNA is introduced in animal

and plant cells respectively are microinjection and gene gun.

(i) Micro-injection: Recombinant DNA is directly injected into the

nucleus of an animal cell.
(ii) Gene gun/Biolistics: This method is suitable for plants. Cells are
bombarded with high velocity micro-particles of gold or tungsten
coated with DNA.
18. Why is “Agrobacterium–mediated genetic engineering
transformation” in plants considered as natural genetic
engineering?
Ans. Agrobacterium tumefaciens is a plant pathogen that infects many
dicot plants. It causes crown gall disease in plants which is induced by Ti
plasmid or the tumour-inducing plasmid. It is able to deliver a piece of
DNA (T–DNA) to transform normal plant cell into a tumor and directs
these tumor cells to produce the chemicals required by pathogen. So if
the gene of interest is inserted in this T-DNA region of Ti plasmid of
Agrobacterium, that will be integrated into the plant genome naturally.
19. What is a bioreactor? Explain stirred-tank bioreactor in brief.

Ans. The bioreactor is a large vessel used to carry out a biological

reaction where raw materials are converted into specific products,
individual enzymes, etc., using microbial, plant, animal or human cells. A
bioreactor provides the optimal conditions for achieving the desired
product by providing optimum growth conditions (temperature, pH,
substrate, salts, vitamins, oxygen).
The most commonly used bioreactors are of stirring type.

A stirred-tank reactor is usually cylindrical or with a curved base to

facilitate the mixing of the reactor contents. The stirrer facilitates even
mixing and oxygen availability throughout the bioreactor. Alternatively,
air can be bubbled through the reactor. The bioreactor has an agitator
system, an oxygen delivery system and a foam control system, a
temperature control system, pH control system and sampling ports so
that small volumes of the culture can be withdrawn periodically.
20.What is the major difference between simple stirred tank bioreactor
and sparged stirred tank bioreactor? What is its advantage? Draw
neat and labelled diagram of sparged stirred tank bioreactor.

Ans. Sterile air bubbles are sparged into the sparged stirred - tank bioreactor.
The surface area for oxygen transfer is increased.

***************************************************************************