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Principles Of Biotechnology
The European Federation of Biotechnology (EFB) has come up with

a new definition that involves both the traditional and the modern view of
biotechnology. It states that
‘Biotechnology is the integration of natural science and organisms, cells, parts

thereof, and molecular analogs for products and services’.
The two core techniques responsible for the birth of modern biotechnology are:
· Genetic Engineering: It involves techniques that change the chemistry of the
genetic material such that when they are introduced into the host, the host
phenotype changes.
· Bioprocess engineering: Maintenance of a contamination-free environment to
enable the growth of only the desired microbe in large quantities during the
manufacture of products like vaccines, antibiotics etc.
Genetic Engineering
The techniques of genetic engineering such as recombinant DNA
technology, gene cloning etc. overcome this problem, allowing us to introduce
only desired genes. Genetically modifying an organism has the following basic
steps:
· Identification of DNA containing desired genes.
· Introduction of desired DNA into the host organism.
· Maintenance of the introduced DNA in the host genome and transfer of the
DNA to its progeny.

Tools Of Biotechnology
Genetic engineering or recombinant DNA technology is possible only if we have
some important tools. The following are some important tools of biotechnology.
1) Restriction Enzymes
Restriction enzymes, also known as ‘restriction endonucleases’ are molecular
scissors that can cut DNA at specific locations. These are part of a larger class of
enzymes called ‘Nucleases’. Nucleases are of two kinds:
· Exonucleases – Enzymes that remove nucleotides from the ends of DNA.
· Endonucleases – Enzymes that make cuts at specific positions in the DNA.
‘Hind II‘ – the first restriction endonuclease to be isolated, identifies a specific six
base pair sequence and always cuts the DNA at a particular point.
This specific sequence is the ‘Recognition sequence‘. Today,
there are more than 900 restriction enzymes, isolated from about 230 bacterial
strains. Each of these enzymes recognizes different recognition sequences.
There is a naming convention to name restriction enzymes. Let’s learn this using
EcoRI as an example:
· Eco – indicates that this enzyme was isolated from Escherichia coli.
· R – refers to the name of the strain.
· I – is the Roman numeral that indicates the order of isolation of this restriction
enzyme from the strain of bacteria.

Mode of Action of Restriction Enzymes
Endonucleases perform their function in the following manner:
· Inspect the length of DNA for the specific recognition sequence.

· Bind the DNA at the recognition sequence.
· Cut the two strands of DNA at specific points in their sugar-phosphate
backbone.
Restriction enzymes recognize specific palindromic nucleotide sequences in the
DNA. A palindrome is a sequence of base pairs that reads the same on both DNA
strands when reading in the same orientation. For example –
5 ′ — — G A A T T C — — 3 ′
3 ′ — — C T T A A G — — 5 ′
Restriction enzymes cut the DNA strand a little away from the center of the
palindromic site, but between the same two bases on both strands.
This gives rise to single-stranded, overhanging stretches on each strand
called ‘sticky ends’. They are called ‘sticky’ because they can bind to their
complementary cut counterparts.
Using restriction enzymes we can generate recombinant DNA that contains DNA
from different sources. Cutting these DNA sources with the same restriction
enzyme gives fragments with the same kind of ‘sticky ends’, that can then be
joined using DNA ligases.
2) Cloning Vectors
Just the way a mosquito acts as a ‘vector’ to transfer the malarial parasite into the
human body, we need vectors to transfer the cut DNA into a host organism.
Vectors are one of the important tools of biotechnology.
Plasmids make good vectors because they can replicate in bacterial cells,
independent of the control of the chromosomal DNA.
The vectors in use currently are engineered such that they help in easy linking
of foreign DNA and allow selection of recombinants over non-recombinants.
A vector needs the following features to enable cloning:
(i) Origin Of Replication (ori)

1. This is the sequence from where replication begins.
2. Linking a piece of DNA to this sequence causes it to replicate in the host cell.
3. This sequence also controls the copy number of the linked DNA. Therefore, the
target DNA needs to be cloned into a vector whose ‘ori’ supports high copy
number, in order to recover large amounts of the DNA.
(ii) Selectable Marker

1. The vector also needs to have a selectable marker which allows the selection of
recombinants over non-recombinants.
2. In terms of E. coli, some useful selectable markers are genes that provide
resistance to antibiotics like ampicillin, kanamycin, chloramphenicol etc.
3. Since the normal E. coli cells do not carry these resistance genes, it becomes
easy to select the recombinants.
(iii) Cloning Sites
In order to attach the foreign DNA to a vector, the vector should have a
recognition site for a specific restriction enzyme.
Multiple recognition sites will result in multiple DNA fragments, complicating
the process of cloning.
A vector has more than one antibiotic resistance gene. The foreign DNA is
ligated into a restriction site in one of the antibiotic resistance genes.
(iv) Vectors to Clone Genes in Plants and Animals

Long before us, bacteria and viruses knew how to transfer genes into plants and
animals.
For example, Agrobacterium tumifaciens, a pathogen on dicot plants, transfers
‘T-DNA’ that transforms normal plants cells into tumours. These tumours then
produce chemicals that the pathogen requires.
With better understanding, we have now converted these pathogens into
useful vectors to deliver genes of interest to the plants or animals.
The main characteristics of pBR322 are:

· Restriction sites: BamH I, Hind III, Sal I, Pvu I, Pvu II, Pst I, EcoR I, Cla I
· Selectable marker: antibiotic resistance genes for ampicillin (ampR) and
tetracycline (tetR)
· ORI: the origin of replication
· ROP: It codes for proteins, which are involved in the process of replication of
plasmid
Recombinant Plasmid
Recombinant plasmids are the plasmids into which a foreign DNA fragment of
gene is inserted. These recombinant plasmids independently replicate from the
chromosomal DNA of the host
.
Cells that contain recombinant plasmids usually are selected by taking to
advantage the antibiotic resistance marker found on the vector plasmid.
The cells containing recombinant plasmids can be recognized as containing
recombinant plasmids by screening for insertional inactivation of a second
genetic marker on the plasmid.
Advantages of Plasmids
1. · Plasmids are used as tools to transfer, manipulate and clone genes

2. · Since bacteria can rapidly divide, it can be used as factories to copy DNA
fragments in large quantities
3. · Using plasmids as vectors is advantageous as it is easy to isolate and manipulate
because of small size
4. · Due to its circular configuration, it is more stable
5. · Replicate independant of the host
3) Competent Host
Host cells are bacterial cells which take up the recombinant
DNA. Since DNA is hydrophilic, it cannot pass through the cell membrane of bacteria
easily.
Therefore, the bacterial cells have to be made ‘competent’ to take up the DNA.
Some procedures that make the cells competent are treatments with a specific
concentration of divalent cation like calcium.
This makes it easy for the DNA to enter the cell wall through pores. Incubation of
cells with the recombinant DNA on the ice, followed by heat shock at 42°C and
another incubation on ice, enables the cells to take up the DNA.
There are several other methods to introduce foreign DNA into host cells.
The ‘microinjection’ method involves injecting the recombinant DNA

directly into the nucleus of an animal cell.
The ‘bolistics’ or ‘gene gun’ method bombards plant cells with high-
velocity micro particles of gold or tungsten coated with DNA.
The last method uses ‘disarmed pathogen vectors’ (discussed above)

to transfer the recombinant DNA into the infected host cells.
BIBLIOGRAPHY
1. Class 12 NCERT textbook
2. Wikipedia (Google)
3. Bank Of Biology

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Principles Of Biotechnology

The European Federation of Biotechnology (EFB) has come up with

‘Biotechnology is the integration of natural science and organisms, cells, parts

· Identification of DNA containing desired genes.

· Introduction of desired DNA into the host organism.

DNA to its progeny.

· R – refers to the name of the strain.

enzyme from the strain of bacteria.

· Inspect the length of DNA for the specific recognition sequence.

(i) Origin Of Replication (ori)

(ii) Selectable Marker

(iv) Vectors to Clone Genes in Plants and Animals

The main characteristics of pBR322 are:

1. · Plasmids are used as tools to transfer, manipulate and clone genes

The ‘microinjection’ method involves injecting the recombinant DNA

The last method uses ‘disarmed pathogen vectors’ (discussed above)

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