Biotechnology Principles and

Processes
• Principles of biotechnology

• Tools of Recombinant DNA technology

- Restriction enzymes
- Cloning vectors
- Competent host

• Processes of Recombinant DNA technology

- Isolation of genetic material
- Cutting of DNA at specific locations
- Amplification
- Insertion
- obtaining gene product
- Downstream processing
BIOTECHNOLOGY
• Biotechnology deals with techniques of using live organisms or their enzymes for
products and processes useful to humans.
• The European Federation of Biotechnology (EFB) defines Biotechnology as ‘the
integration of natural science & organisms, cells, parts thereof, & molecular
analogues for products & services.
• Biotechnology deals with:
- In vitro fertilisation (‘test-tube’ baby programme)
- Synthesis and using of a gene
- Preparation of a DNA vaccine
- Correcting a defective gene
A - Principles of BT
• The two core techniques of modern biotechnology are:

a. Genetic engineering
• genetic material (DNA & RNA) is chemically altered and introduced into host
organisms to change phenotype of host.
b. Bioprocess engineering
• Maintenance of sterile (microbial contamination-free) ambience in chemical
engineering processes to enable growth of only the desired microbe/eukaryotic
cell in large quantities for manufacture of - antibiotics, vaccines, enzymes, etc.
Principles of BT
• Traditional hybridization- inclusion and multiplication of undesirable genes along
with desired genes.
• Genetic engineering - isolate and introduce only desirable genes into the target
organism.
• A piece of DNA is not able to multiply itself in the progeny cells of the organism.
But, when it gets integrated into the recipient genome, it multiplies and inherits
along with the host DNA.
• Recombinant DNA- use of gene cloning and gene transfer, to isolate and
introduce desirable genes without introducing undesirable genes into the target
organism.
Principles of BT
• Origin of replication - specific DNA sequence in a chromosome which is
responsible for initiating replication
• Foreign DNA must be part of a chromosome(s) which has “Ori” for it to get
replicated.
• Cloning - making multiple identical copies of any template DNA
• Plasmid - autonomously replicating circular extra-chromosomal DNA, used as
vector.
• Restriction Enzymes (‘molecular scissors’) - enzymes that cut DNA at specific
locations
• DNA ligase - acts on cut DNA molecules and joins their ends
Principles of BT
• First recombinant DNA - Stanley Cohen & Herbert Boyer (1972)
• Linked a gene of antibiotic resistance with a native plasmid of Salmonella
typhimurium.
• This DNA is transferred into Escherichia coli – plasmid replicate using new host’s
DNA polymerase enzyme & makes multiple copies
• Cloning of antibiotic resistance gene in E. coli. - Ability to multiply copies of
antibiotic resistance gene in E. coli
Principles of BT
• 3 basic steps in genetically modifying an organism:
a) Identification of DNA with desirable genes
b) Introduction of the identified DNA into the host
c) Maintenance of introduced DNA in the host and transfer of the DNA to its
progeny
Tools of Recombinant DNA technology
- Restriction enzymes
- Cloning vectors
- Competent host
1) Restriction enzymes
• In 1963, two enzymes restricting growth of bacteriophage in E. coli were isolated:
- added methyl groups to DNA
- restriction endonuclease cut DNA.
• RE - Hind II - cuts DNA molecules at a particular point by recognizing a specific
sequence of six base pairs called - recognition sequence for Hind II.
• Today over 900 RE isolated from over 230 strains of bacteria.
• Naming of the RE - First letter genus and the second two letters indicate species
of the prokaryotic cell from which they were isolated. E.g. EcoRI from E. coli RY
13 (R = strain, Roman numbers = order in which enzymes were isolated from that
strain of bacteria).
1) Restriction enzymes
• Restriction enzymes belong to a class of enzymes called nucleases. 2 types
exonucleases & endonucleases.
• Exonucleases - remove nucleotides from the ends of the DNA.
• Endonucleases - cut at specific positions within the DNA.

• RE - binds to specific recognition sequence of the DNA and cut each of the two
strands at specific points in their sugar-phosphate backbones (phosphodiester bonds in
DNA/RNA)
• RE - recognizes a specific palindromic nucleotide sequences in the DNA i.e. - a
sequence of base pairs that reads same on the two strands in 5' → 3' direction
and in 3' → 5' direction. E.g 5' —— GAATTC —— 3’
3' —— CTTAAG —— 5'
1) Restriction enzymes
• RE- cuts strand little away from centre of palindrome sites, but between same
two bases on the opposite strands – produces single stranded overhanging
stretches at the ends called sticky ends.
• They form H-bonds with their complementary cut counterparts. This stickiness
facilitates action of the enzyme DNA ligase
• When 2 DNA strands are cut by same RE- resultant DNA fragments have same
kind of sticky-ends and these are joined together by DNA ligases.
1) Restriction enzymes
• Separation and isolation of DNA fragments: - DNA fragments formed by RE are
separated by - technique gel electrophoresis.
• DNA fragments are negatively charged- can be separated by moving them
towards the anode under an electric field through a medium/matrix such as
agarose (a natural polymer extracted from sea weeds)
• DNA fragments separate (resolve) according to their size through sieving effect
provided by the agarose gel.
• Smaller sized fragment move farther.
1) Restriction enzymes
• Separation and isolation of DNA fragments:
• Separated DNA fragments can be visualized - staining with ethidium bromide
followed by exposure to UV radiation.
• Bright orange coloured DNA bands can be seen.
• Separated DNA bands are cut out from agarose gel and extracted from gel piece.
This step is called elution.
• These purified DNA fragments are used in constructing recombinant DNA by
joining them with cloning vectors.
2) Cloning vectors
• They are the DNA molecules that can carry a foreign DNA segment and replicate
inside the host cells.
E.g. Plasmids and bacteriophages.
• Bacteriophages (high number per cell) have very high copy numbers of their
genome within the bacterial cells.
• Some plasmids have only 1-2 copies per cell. Others may have 15-100 copies per
cell.
• When the cloning vectors are multiplied in the host the linked piece of DNA is
also multiplied to the numbers equal to the copy number of the vectors.
2) Cloning vectors - Features
a) Origin of replication (ori): sequence from where replication starts. A piece of
DNA linked to ori can replicate within the host cells. This also controls the copy
number of the linked DNA.
• For getting many copies of the target DNA it should be cloned in a vector whose
origin supports high copy number.
2) Cloning vectors - Features
b) Selectable marker (marker gene): helps to select transformants and eliminate the
non-transformants.
• Transformation - a piece of DNA is introduced in a host bacterium.
• Selectable markers of E. coli - genes coding resistance to antibiotics like
ampicillin, chloramphenicol, tetracycline or kanamycin, etc.
• Normal E. coli cells do not carry resistance against any of these antibiotics
2) Cloning vectors - Features
c) Cloning sites: to link the alien DNA, the vector needs very few recognition sites
for restriction enzymes.
• If more than one recognition sites in vector – generates several fragments -
complicates the gene cloning.
• Ligation of alien DNA - at restriction site present in one of the two antibiotic
resistance genes.
• E.g. ligation of a foreign DNA at the Bam H I site of tetracycline resistance gene in
the vector pBR322.
2) Cloning vectors - Features
• Foreign DNA + vector Pbr322 = transformants/recombinants + non-recombinants
• Transformants – lost tetracycline resistance
• Non-recombinants – have tetracycline resistance +ampicilin resistance
• Recombinants will grow only in ampicillin medium not tetracycline medium.
• Non-recombinants will grow on the medium containing both the antibiotics
• simultaneous plating on 2 plates having different antibiotics required – difficult.
2) Cloning vectors - Features
• Easier method - differentiate recombinants from non-recombinants on the basis
of their ability to produce colour in presence of a chromogenic substrate.
• A recombinant DNA inserted within coding sequence of an enzyme, α-
galactosidase = enzyme inactivated. It is called insertional inactivation
• Recombinant colonies - do not produce any colour.
• Non-recombinant bacteria - give blue coloured colonies in presence of
chromogenic substrate.
2) Cloning vectors - Features
d) Vectors for cloning genes in plants and animals
• pathogens can be transformed into useful vectors for delivering genes to plants
& animals.
• E.g. Agrobacterioum tumifaciens (pathogen of many dicot plants) can deliver a
piece of DNA (T-DNA) to transform normal plant cells into a tumor - produce the
chemicals required by the pathogen.
• Tumor inducing (Ti) plasmid of A. tumifaciens modified into a cloning vector - not
pathogenic, helps deliver genes of interest into plants.
• Retroviruses in animals can transform normal cells into cancerous cells. Are used
to deliver desirable genes into animal cells.
3) Competent host
Method 1 - to introduce alien DNA into host cells
• DNA - hydrophilic molecule- cannot pass through cell membranes.
• So, bacterial cells are treated with specific concentration of divalent cation (e.g.
calcium) – then DNA enters the bacterium through pores in cell wall.
• Cells are incubated with recombinant DNA on ice.
• Then given heat shock- placing briefly at 420C
• Then back on ice.
• This enables the bacteria to take up recombinant DNA.
3) Competent host
Other Methods - to introduce alien DNA into host cells
• Micro-injection: recombinant DNA is directly injected into the nucleus of an
animal cell.
• Biolistics (gene gun): cells are bombarded with high velocity micro-particles of
gold or tungsten coated with DNA. This method is suitable for plants.
• ‘Disarmed pathogen’ vectors: which when infect the cell, transfer the
recombinant DNA into the host.
B - Process of BT
The steps involved are:

1. Isolation of the Genetic Material (DNA)

2. Cutting of DNA at Specific Locations
3. Amplification of Gene of Interest using PCR
4. Insertion of Recombinant DNA into the Host Cell/Organism
5. Obtaining the Foreign Gene Product
6. Downstream Processing
1) Isolation of the Genetic Material
• Pure DNA (free from other macro-molecules) - bacterial cells/plant or animal
tissue - treated with enzymes -lysozyme (bacteria), cellulase (plant cells),
chitinase (fungus) etc.
• Cell is broken to release DNA along with other macromolecules (RNA, proteins,
polysaccharides and lipids).
• Genes (DNA) are intertwined with proteins such as histones.
• RNA is removed by treating with ribonuclease.
• Proteins are removed by treatment with protease.
• Other molecules are removed by appropriate treatments.
• When chilled ethanol is added purified DNA precipitates out as a collection of
fine threads in the suspension.
2) Cutting of DNA at Specific Locations
• Restriction enzyme digestions are performed by incubating purified DNA with the
restriction enzyme, at the optimal conditions.
• Agarose gel electrophoresis is employed to check the progression of a restriction
enzyme digestion. As DNA is negatively charged, it moves towards the anode.
The process is repeated with the vector DNA also.
• After cutting the source DNA and the vector DNA, the cut out gene (DNA
segment) of interest from the source DNA and the cut vector are mixed and
ligase is added.
• This creates recombinant DNA.
3) Amplification of Gene of Interest using PCR

• Polymerase Chain Reaction (PCR) is the synthesis of multiple copies of the gene
of interest in vitro using 2 sets of primers & the enzyme DNA polymerase.
• Primers are small chemically synthesized oligonucleotides that are
complementary to the regions of DNA.
• The enzyme extends the primers using the nucleotides and the genomic DNA
(template). Through continuous DNA replication, the DNA segment is amplified
up to 1 billion copies.
• For amplification a thermostable DNA polymerase (isolated from a bacterium,
Thermus aquaticus) is used.
3) Amplification of Gene of Interest using PCR

• It remains active in high temperature during the denaturation of double stranded

DNA.
• The amplified fragment can be used to ligate with a vector for further cloning.
4) Insertion of Recombinant DNA into the Host
Cell/Organism
• There are several methods of introducing the ligated DNA into recipient cells.
Recipient cells take up DNA present in its surrounding.
• If a recombinant DNA bearing ampicillin resistant gene (a selectable marker
gene) is transferred into E. coli cells, the host cells become ampicillin-resistant
cells.
• If the transformed cells are spread on agar plates containing ampicillin, only
transformants will grow, untransformed recipient cells will die -
• Ampicillin resistance gene is selectable marker.
5) Obtaining the Foreign Gene Product
• Ultimate aim of recombinant DNA technology - produce a desirable protein. The
foreign gene gets expressed under appropriate conditions.
• If a protein encoding gene is expressed in a heterologous host, it is called a
recombinant protein.

Method 1
• Cells with foreign genes - grown on a small scale in laboratory.
• Cultures - used to extract desired protein and purify it by using different
separation techniques.
5) Obtaining the Foreign Gene Product
Method 2
• Cells - multiplied in a continuous culture system.
• Used medium is drained out from one side while fresh medium is added from the
other.
• It maintains the cells more physiologically active and so produces a larger
biomass leading to higher yields of desired protein.
Bioreactors
• for producing large quantities of products, large volumes (100-1000 litres) of
culture can be processed.
• Bioreactors - vessels in which raw materials are biologically converted into
specific products, enzymes etc., using microbial plant, animal or human cells.
• Provides the optimal growth conditions (temperature, pH, substrate, salts,
vitamins, oxygen) for achieving the desired product.
• Most common bioreactors are of stirring type (stirred-tank reactor).
• Usually cylindrical or with a curved base to facilitate mixing of the reactor
contents. Stirrer facilitates even mixing and oxygen availability. Alternatively air
can be bubbled through the reactor.
Bioreactors
• The bioreactor has
- An agitator system
- An oxygen delivery system
- A foam control system
- A temperature control system
- pH control system
- Sampling ports (for periodic withdrawal of the culture).
6. Downstream Processing
• It is a series of processes - separation and purification of products after the
biosynthetic stage – before marketing the product
• The product is formulated with suitable preservatives.
• Formulation require clinical trials in case of drugs.
• Strict quality control testing for each product is also required.
• Downstream processing and quality control testing vary from product to product.