Chapter 11

Biotechnology: Principles and Processes

Biotechnology:
 It can be defined as use of microorganisms, plants and animals cells or their components to produce
products and processes useful to humans.

 According to European Federation Technology (EFT) biotechnology is the integration of natural

science and organisms, cell, parts thereof and Molecular analogues for products and services.

 The term biotechnology was given by Karl Ereky.

Principles of Biotechnology:
i. Genetic engineering: it refers to artificial synthesis, isolation, modification, combination, addition and
repair of genetic materials i.e. DNA / RNA to alter the phenotype of host organism to suit human needs.

ii. Chemical engineering: adequate maintenance of a sterile conditions to support growth of desired
microbes / eukaryotic cells in large quantities for manufacture of biotechnological products like
antibiotics, vaccines and enzymes etc.

Technique of genetic engineering / Recombinant DNA technology:

i. Creation of Recombinant DNA by combining desired genes.
ii. Gene transfer
iii. Maintenance of DNA in host and gene cloning.

Fate of alien DNA in host cell:

 The alien / foreign piece of DNA would not be able to multiply itself in the progeny cells of the
organism. But when it gets integrated into the genome of the recipient, it may multiply and be
inherited along with the host DNA. This is because the alien piece of DNA has become part of a
chromosome, which has the ability to replicate.
 In a chromosome, there is a specific DNA sequence called origin of replication that is site of
initiation of DNA replication which must be needed for the alien DNA to be integrated with the host
DNA and multiply normally.
 This process of replication of alien DNA in the host organism itself by getting integrated with the
host / recipient DNA at a specific ori sequence is called cloning (making multiple identical copies of
any template DNA).

Construction of first Recombinant DNA:

It was done by Stanley Cohen and Herbert Boyer in 1972- isolated antibiotic resistance change from plasmid
of bacterium Salmonella typhimurium.

 Isolated the antibiotic resistance gene by cutting the desired piece of DNA from the plasmid.
 Cutting a piece of DNA from a plasmid, with the help of restriction endonuclease / molecular scissor.
 The piece of DNA cut from the plasmid was then linked with the plasmid DNA, with the help of
DNA ligase enzyme.
  This newly formed DNA having integrated fragment of antibiotic resistant gene is called
Recombinant DNA (rDNA).
 When this DNA is transferred into E. coli, it could replicate using the new host's DNA polymerase
enzyme and make multiple copies i.e. cloning.

Tools of Recombinant DNA Technology:

Three Types of biological tools are used in the synthesis of Recombinant DNA;
1. Enzymes
2. Cloning vectors
3. Competent host organism

ENZYMES:
Many types of enzymes are used in genetic engineering like cleaving enzymes and joining enzymes etc.

Cleaving enzymes: the help in cutting the DNA. These are- exonuclease, endonuclease and restriction
endonuclease.
i. Exonuclease: cut of DNA nucleotides from ends.
ii. Endonuclease: cut DNA at any point except the ends.
iii. Restriction Endonuclease: cut DNA duplex at specific points in such a way that single-stranded
free ends project from each strand of DNA duplex. this free and circle sticky ends.

Joining enzymes: these are ligases/ DNA ligase. These are also called molecular glues as they join the two
fragments of cleaved DNA.

Restriction Endonuclease / molecular Scissors:

 Each restriction endonuclease recognise is a specific palindromic nucleotide sequence in the DNA.
 Palindrome in DNA is a sequence of base pairs that reads same order in forward (5' to 3') or backward
(5' to 3') direction.
For example;
5' - GAATTC - 3'
3' - CTTAAG - 5'

Nomenclature of restriction enzymes:

i. The name of the enzyme is based on the microbe from which it is obtained.
ii. The first letter of the enzyme should be capital and it is the first letter of the genus of the microbe.
iii. Two letters of the enzyme denotes the starting two letters of the species of the microbe and it should
be in small letters.
 iv. Fourth letter of enzyme is the first letter of the strain of the microbe. it can be small or capital, based
on the strain type.
v. The Roman number written at the end of the name indicates the order in which of the enzyme was
isolated from the strain of the prokaryotic cell.

For example;
i. Escherichia coli RY13 Eco R1
ii. Haemophilus influenzae Rd Hind II
iii. Escherichia coli R245 Eco RII

Hind II: it is the first isolated restriction endonuclease isolated from Haemophilus influenzae. It always cut
DNA molecule at a particular point by recognising a specific sequence of 6 base pairs and known as
recognition sequence.

Mechanism of action of restriction endonucleases:

 Restriction enzymes cut the strand of DNA little away from the centre of the palindrome sides but
between the same two bases on the opposite strands.
 This leaves single stranded portions at the ends called sticky ends /overhanging stretches. They are
named so because they form hydrogen bonds with complementary strands.
 The stickiness of the ends facilitates the action of the enzyme DNA ligase.
 These sticky ends are complementary to each other when cut by same restriction enzyme, therefore they
can be joined together end to end by using DNA ligase.

Separation and isolation of DNA fragments:

 The molecule of DNA can be cut into fragments by the use of enzyme restriction endonuclease. These
fragments are separated by using gel electrophoresis (technique used for the separation of substances of
different ionic properties and size).
 Since DNA fragments are negatively charged they will move towards the anode which is positively
charged, of the Apparatus through the medium / matrix- agarose (natural Polymers / polysaccharide
extracted from seaweeds) under the influence of electric field applied.
 The smaller fragments move farther away as compared to larger fragments.
 Now, the DNA fragments are strained with its Ethridium Bromide for visualization.
 The bright orange colour DNA bands can be seen after the exposure of UV light.
 These bands, then are cut out from the gel and extracted by using a convenient technique. This step is
called Elution.
 DNA fragments are then purified and then used for construction of Recombinant DNA.

CLONING VECTORS/ VEHICLE DNA:

These are the DNA molecules which carry foreign DNA segment into the host cell for the formation of
Recombinant DNA. These are also known as vehicle DNA or Gene carriers.

Copy number: it can be defined as the number of copies of factor present in a DNA. So in Recombinant
DNA technique, those vectors are selected which have high copy number. It is measured as copies per cell.

Major vectors used in Recombinant DNA technology are:

1. Plasmids: these are autonomously replicating circular extrachromosomal DNA.

For example; pBR322, where p, B and R stands for plasmid, Boliver and Rodriquez. This is the first
Artificial cloning vector.
Plasmids only have 1-2 copies per cell.

2. Bacteriophages: these are the viruses that infect bacteria. The copy number is very high that's why it
is used as cloning vector.

3. Cosmids: they are hybrid vectors derived from classmates which contain a cos site of Lambda
phage.

**If the alien piece of DNA is linked with cloning vectors the, it's a number can be multiplied equal to the
copy number of that cloning vector.

Features required facilitating cloning into vectors:

1. Origin of replication:
A plasmid should have a particular sequence from where replication get started called ori / origin of
replication. Any piece of DNA when linked to this sequence can be made to replicate within the host cells.
This property is used to take a number of copies of linked DNA. It is desirable that cloning of target DNA
should be carried out in such a vector where origin facilities high copy number.

2. Selectable marker:
They help in identifying and eliminating non transformants and selectively permit the growth of the
transformants. Those host cells which have got Recombinant DNA are known as transformants while those
have not got Recombinant DNA are known as non transformants. These genes encoding two antibiotics such
as Ampicillin (amp R), Tetracycline (tet R), chloramphenicol etc. are selectable markers for E coli.

3. Recognition sites / cloning sites:

At this site, the restriction endonuclease enzyme makes a cut so that the alien DNA segment may be joined
to the vector. Only one restriction site is preferable for an enzyme, so that only one cut is induced in the
plasmid to avoid complications in gene cloning.

Ligation of alien DNA:

 It takes place at a restriction site present in one of the two antibiotic resistant genes. For example, a
foreign DNA can be ligated at the Bam HI site of tetracycline resistant gene in E coli cloning vector
pBR 322.
 The insertion of alien DNA results in the loss of tetracycline resistance by the Recombinant plasmid.
 Now the recombinant will not grow in a medium containing tetracycline antibiotic but the non-
recombinant will grow and both will grow in antibiotic ampicillin.
 That’s how they can be separated and selected.

Selection of recombinants:
 Selection of recombinants due to inactivation of antibiotics is a cumbersome process because it
requires simultaneous plating on two plates having different antibiotics. So alternative selectable
markers are developed which differentiate recombinants and non Recombinant on the basis of their
ability to produce colour in the presence of chromogenic substrate.
 In this method (colour reaction method) and Recombinant DNA is integrated in the coding sequence
of an enzyme, beta galactosidase, so that enzyme gets inactivated. This is called insertional
inactivation. Now,recombinants will show no colour in the presence of chromogenic substrate unlike
the non Recombinant which give blue colour in the presence of chromogenic substrate.

4. Vectors for cloning genes in plants and animals:

Soil contains a bacterium- Agrobacterium tumefaciens, which infects broad leaved plants. It's plasmid
contains T-DNA (segment of DNA which causes tumor in plant cells) which when integrated with the
chromosomal DNA of the plant cells, they undergo proliferation and causes tumor. Therefore this plasmid is
called Ti plasmid (Ti = tumor inducing).
 Since, gene transfer occurs without human effort, this bacterium is known as “Natural genetic
engineer of plants”.
 Now plant biologics use this Ti plasmid but after eliminating T-DNA from the Ti-plasmid. The
transformed bacteria do not cause diseases.

Retrovirus, adenovirus, papillomavirus are also used as cloning vectors in animal cells.

COMPETENT HOST ORGANISM: (For transformation with Recombinant DNA)

DNA being hydrophilic molecule can not cross directly through the cell membrane. Therefore, certain
treatments are made in the host cell to become competent to take up DNA. These are as follows;

1. Chemical methods:In this method, the cell is treated with a specific concentration of divalent cation,
Ca2+, 20% polyethylene glycol (PEG), is added to it, that increases pore size in the cell. The cells are
then incubated with Recombinant DNA on ice, followed by placing them briefly at 42°C and then
putting back on ice. This is called heat shock. Because of this permeability of bacterial (host) cell
increases.
2. Electroporation: It refers to the introduction of DNA into plant cells by making minute pores in the
plant cell membrane by applying suitable voltage to the suspension of plant protoplast in suitable ionic
solutions containing linearized Recombinant plasmid DNA.

3. Particle bombardment gun method: This technique is also known as a biolistic technique. This
technique involves shooting foreign DNA into plant cells at a very high speed (1400 ft/sec).In this; the
foreign DNA is coated on gold or tungsten particles. These microparticles of about 1-2 micrometer are
shooted with the help of gene guns. This method is mostly used in plant cells.

4. Micro injection: In this method, the DNA solution is directly injected into the nucleus of animal cells
using capillary class micropipettes or micro injections. This is most common in case of animal cells.

Processing of DNA Recombinant Technology:

1. Isolation of genetic material:
 The DNA is isolated first which involves following steps; envelopes are digested first by using
certain enzymes like lysozyme for bacterial cells, cellulase for plant cells and chitinase for fungi
cells.
 The next step is purification of DNA mixed with RNA or other proteins like histones. Hence, our
RNAs and proteases are used to purify DNA.
 Finally, the pure DNA molecules are precipitate it out of by adding chilled ethanol and collected in
the suspension.

2. Cutting of DNA at specific location:

 It is done by using restriction enzymes.
 The purified DNA is incubated with the specific restriction enzyme at conditions optimum for the
enzyme to act.
3. Isolation of desired DNA fragments: it is done by using agarose gel electrophoresis technique which
has already been discussed previously.

4. Amplification of gene of interest using PCR:

Polymerase chain reaction is a technique of synthesizing multiple copies of the desired genes a DNA in
vitro. It was developed by Karry Mullis.

Requirements of PCR:

1. DNA template- the desired segment of the target DNA that is to be amplified.
2. Primers- these are oligonucleotides of 10 to 8 nucleotides that are complementary to DNA template
region.
3. Enzyme- high temperature (>90°C) stable DNA polymerase ( usually Taq Polymerase) is used for
synthesis of new DNA molecule required.

**Taq polymerase is obtained from Thermusaquaticus bacterium.

Mechanism of PCR:

1. Denaturation- target DNA is heated to high temperature of about 96°C for 1 minute. This results in
the separation of the two strands of the target double stranded DNA.

2. Annealing- it is carried out by Primers, which are added in the reaction. Primers aneal or join to the
three prime and of each separated strength. It occurs at 54°C or 30 seconds.

3. Extension- it is done by DNA polymerase by adding nucleotides complementary to the template in

the reaction. It occurs at 72°C for 1 minute in the presence of dNTPs and Mg2+.

These steps are repeated for many times to obtain several copies of the desired DNA.
5. Ligation of DNA fragments into a vector:
The source DNA / gene of interest is ligated with the vector DNA (cloning) with the help of enzyme DNA
ligase to form Recombinant DNA.

6. Insertion of Recombinant DNA into host cell / organism:

 Once a cell receives a foreign DNA fragment and becomes transformed into a genetically modified
cell it starts behaving differently.
 For example, if a Recombinant DNA bearing antibiotic ampicillin resistant gene is inserted in E coli
cells the bacteria becomes resistant to ampicillin. If such bacteria are transferred on culture plate
containing ampicillin, only resistant forms will grow. And otthers will die. This method is used to
separate transformants and non transformants. The gene responsible for ampicillin resistance is
known as selectable marker.
 The cell containing the foreign DNA or gene is cultured on an appropriate medium at optimum
conditions. The DNA gets multiplied.

7. Obtaining the foreign gene product:

 A protein encoding genes expressed in a heterologous host is called Recombinant protein. Cells
having genes of interest can be grown on a small or on a large scale.
 Small scale production can be done through culturing in laboratory while large scale production is
done by using bioreactors.

BIOREACTORS:

These are large volume (100-1000L) vessels in which raw materials are biologically converted into specific
products, individual enzymes, etc. using microbial plants animals or human cells.

It provides optimum conditions for achieving the desired product by providing optimum growth conditions
like temperature, pH, salt, substrate, vitamins and oxygen.

The most commonly used bioreactors are of stirring type:

1. Simple stirred tank bioreactor: these are cylindrical shaped and facilities mixing of reactor contents and
checks oxygen availability throughout the bioreactor.

2. Sparged stirred tank bioreactor: stirred tank bioreactor, sterile air Bubbles are passed, to increase the
surface area for oxygen transfer.
Components of bioreactor:
*Agitator system
*Oxygen delivery system
*Foam control system
*Temperature and pH control system
*Sampling ports to withdraw small volumes of culture

8. Downstream processing:

After the product formation in bioreactors, they are subjected to a series of processes, collectively called
downstream processes. This includes,
 Separation of desired products
 Verification of products
 Formulation with preservatives

These products undergo strict quality control testing before being marketed as finished products.