Biotechnology
Principles of Biotechnology
LECTURE 1
Seep Pahuja
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Biotechnology
Principles of Biotechnology
LECTURE 1
Biotechnology
Biotechnology
➔ In its purest form, the term biotechnology refers to the use of living organisms or
their products to modify human health and the human environment.
Biotechnology
Biotechnology
➔ Nowadays, it is used in restricted sense.
➔ It refers to those processes which includes formation of Genetically modified
Organisms.
Biotechnology
Biotechnology
➔ Technique of using live organisms, their cellular components or enzymes to
produce products and processes useful to us in industry, Agriculture, Medicine and
food processing.
Biotechnology
Classical / Traditional Biotechnology
➔ In includes the process that are based on the natural capabilities of
microorganisms.
➔ Curd, vinegar, ghee, wine and beer and other alcoholic beverages, idli, dosa,
paneer and some other food have been produced using traditional biotechnology.
Biotechnology
Modern Biotechnology
➔ In-vitro fertilisation leading to a ‘test-tube’ baby, synthesising a gene and using it,
developing a DNA vaccine or correcting a defective gene, are all part of
biotechnology.
Biotechnology
Biotechnology
➔ The European Federation of Traditional View
Biotechnology (EFB) has given the
definition of biotechnology which
includes both Modern Molecular Biotechnology
➔ The integration of natural science and organism, cells, parts thereof, and
molecular analogues for products and services.
Biotechnology
Principles of Biotechnology
➔ Main techniques that helped in combining genetic elements of living cells and that
gave birth to Modern Biotechnology are:
Genetic Engineering
Bioprocess Engineering
Biotechnology
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.
Biotechnology
Bioprocess Engineering
➔ Maintenance of sterile (microbial contamination-free) ambience in chemical
engineering processes to enable growth only the desired microbe/eukaryotic cell
in large quantities for the manufacture of biotechnological products like antibiotics,
vaccines, enzymes, etc.
Biotechnology
Gene Cloning
➔ Cloning is making multiple identical copies of any template DNA.
➔ It includes:
Creation of recombinant DNA
Use of gene cloning
Gene transfer
Gene Cloning
Biotechnology
Construction of First Recombinant DNA
molecule
➔ The first recombinant DNA was constructed by
Stanley Cohen and Herbert Boyer in 1972.
Biotechnology
Construction of First Recombinant DNA
molecule
➔ Antibiotic resistance gene was isolated from a plasmid Salmonella typhimurium.
Biotechnology
Construction of First Recombinant DNA
molecule
➔ The cut piece of DNA was then linked with the plasmid DNA.
➔ Recombinant DNA was thus created in vitro.
Biotechnology
Construction of First Recombinant DNA
molecule
➔ When this DNA was transferred into Escherichia coli, it could replicate using new
host’s DNA polymerase enzyme and make multiple copies and also confer
antibiotic resistance to E.coli.
Biotechnology
Basic Steps for creating GMOs
➔ Identification of DNA with Desirable genes.
➔ Introduction of the identified DNA into the host.
➔ Maintenance of introduced DNA in the host and transfer of the DNA to its
progeny.
Biotechnology
Tools for Recombinant DNA Technology
Three types of Biological tools are required for the formation of Recombinant DNA.
Enzymes Cloning vector Competent Host
Restriction enzymes
Polymerase enzymes
Ligase enzymes
Biotechnology
Restriction Enzymes
➔ These were isolated for the first time by W.Arber in 1962 in bacteria.
➔ In 1972, Arber, Smith and Nathan were awarded Nobel prize for the discovery of
Restriction endonucleases.
Biotechnology
Restriction Enzymes
➔ Restriction enzymes are produced by bacteria as a defence mechanism against
infection by bacteriophage.
Biotechnology
Restriction Enzymes
➔ They restrict or prevent viral infection by degrading the DNA of invading viruses.
➔ A restriction enzyme recognizes and binds to DNA at a specific nucleotide
sequence called a recognition sequence or restriction site.
Biotechnology
Restriction Enzymes
➔ The enzyme then cuts both strands of the DNA within that sequence by cleaving
the phosphodiester backbone of DNA.
➔ As they cut the molecule of DNA into desired size, Therefore, they are called as
Molecular Scissors/Chemical Knives/Chemical Scalpel.
Biotechnology
Restriction Enzymes
➔ The DNA of bacterial cell is protected from the cell’s own restriction enzymes by
the addition of methyl groups (-CH3) to adenines or cytosines within the
sequences recognized by the enzymes.
Biotechnology
➔ Restriction enzyme belongs to a larger class of enzymes called Nucleases.
Nucleases
Exonucleases remove Endonucleases which
nucleotides from the end break internal
of a DNA molecule. phosphodiester bonds.
Biotechnology
Biotechnology
Restriction Enzymes
➔ The first restriction endonuclease - Hind II always always cut DNA molecules at a
particular point by recognizing a specific sequence of six base pairs.
5’ GT (Pyrimidine : T or C) (Purine : A or G) AC3’
3’ CA (Purine : A or G) (Pyrimidine : T or C) TG5’
➔ Besides Hind II, we know more than 900 restriction enzymes that have been
isolated from over 230 strains of bacteria each of which recognise different
recognition sequences.
Biotechnology
Recognition Sequence
➔ Each restriction enzyme recognises a specific palindromic nucleotide sequence in
DNA.
➔ A palindrome is a word, phrase, number or other sequence of units that can be
read the same way in either direction.
➔ Palindrome: the nucleotide sequence reads the same on both strands of the DNA
when read in the 5’ to 3’ direction.
5’ 一 GAATTC 一 3’
3’ 一 CTTAAG - 5’
Biotechnology
Recognition Sequence
Biotechnology
Naming of Restriction Enzymes
➔ They are named on the organism from which they were discovered.
◆ First letter - Name of the genus
◆ Second two letters - Species of the prokaryotic cell
Biotechnology
Naming of Restriction Enzymes
Hind II
Haemophilus influenzae Strain d II enzyme
Biotechnology
Naming of Restriction Enzymes
E.g.
➔ EcoRI comes from Escherichia coli
R713.
➔ In EcoRI, the letter ‘R’ is derived
from the name of stain.
➔ BamHI is from Bacillus
amyloliquefaciens (Strain H).
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