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CHAPTER 11
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BIOTECHNOLOGY - PRINCIPLE AND PROCESSES

Important Terms
1) Biotechnology The use of microorganisms, plants, animal cells or their enzymes to
produce products and processes useful to humans.
2) Genetic Engineering Techniques to alter the chemistry of genetic material, DNA and
RNA, to introduce these into host organisms and thus change the phenotype of the host
organism.
3) Gene Cloning Technique of obtaining identical copies of a particular DNA segment or
gene.
4) Plasmid- Autonomously replicating, circular, extra chromosomal DNA in a bacterial cell.
5) Recombinant DNA DNA formed from combining DNA sequences from two different
organisms.
6)

Palindromes Groups of letters that form the same word when read both forward and
backward. (Used for sequence of bases in this chapter)

7) Electroporation Technique by which holes are created in the plasma membrane of a

host cell to facilitate entry of a foreign DNA.
8) Gene therapy Technique of replacement or alteration of a defective gene responsible
for a hereditary disease.
9) Origin of replication Specific DNA sequence from where replication starts.

Two core techniques that gave rise to biotechnology are:

(a) Genetic Engineering
(b) Maintenance of sterile ambience in chemical engineering processes so that
growth of only desired cell takes place.

The construction of the first recombinant DNA was done by Stanley Cohen and
Herbert Boyer in 1972. They linked gene encoding antibiotic resistance to native
plasmid of Salmonella typhionurium.

Tools of Recombinant DNA Technology

(1) Restriction Endonuclease - These are called molecular scissors.

Each restriction endonuclease recognizes a specific palindromic nucleotide sequence.

When cut by the same restriction enzyme, the resultant DNA have the same kind of
sticky ends and these can be joined together using DNA ligases.

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Steps in formation of recombinant DNA by action of restriction endonuclease enzyme

EcoRI

Separation and isolation of DNA fragments is done by a technique known as gel

electrophoresis.

As DNA fragments are negatively charged, they can be separated by forcing them
to move towards the anode under an electric field through a matrix.

2) Cloning vectors To facilitate cloning into a vector, the following features are required:
(a) Origin of replication - The sequence from where replication starts.
(b) Selectable marker It helps in identifying and eliminating non-transformants and
selectively permitting the growth of transformants.
(c) Cloning sites To link the alien DNA, the vector needs to have very few preferably
single, recognition sites for the commonly used restriction enzymes.

E. coli cloning vector pBR322 showing restriction sites.

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(d) Vectors for cloning genes in plants and animals - Agrobacterium tumefaciens is a pathogen
of several dicot plants. It is able to deliver genes to transform eukaryotic cells. It is able
to pass a piece of DNA known as T-DNA to transform normal plant cells into a tumor and
direct these tumor cells to produce chemicals required by the pathogen.

Competent host

A host cell should be competent enough to take the DNA molecule for the transformation.
Following methods are used (i) Using divalent cations
(ii) Heat and cold treatment Cells can be incubated with rDNA on ice and then briefly
placed at 42o C and then putting them back on ice
(iii) Microinjection
(iv) Biolistics or gene gun.

Process of Recombinant DNA technology

(i) Isolation of genetic material Treating the bacterial cell or any other cell with enzymes
such as lysozyme for bacteria, cellulase for plant cell, chitinase for fungal cells. RNA to
be removed by treating with ribonuclease and finally DNA can be precipitated by treating
with chilled ethanol.
(ii) Use of restriction enzymes to cut DNA.
(iii) Amplification of gene using PCR (Polymerase Chain Reaction) technique.

Polymerase chain reaction (PCR)

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(iv) Insertion of Recombinant DNA into the host cell or organism.

(v) Obtaining the foreign gene product - If any protein encoding gene is expressed in a
heterologous host, it is called a recombinant protein.

Cells can be multiplied in a continuous culture system wherein used medium is

drained out from one side while fresh medium is added from the other.

To produce large quantities of products, bioreactors are used where 100-1000 litres
of culture can be processed.

The most commonly used bioreactors are of stirring type.

a) Simple stirred-tank bioreactor

b) Sparged stirred-tank bioreactor through which sterile air bubbles are
sparged

Downstream processing are processes which include separation and purification of

the product before it is ready for marketing as finished product.