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1. Biotechnology Process

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Kritika Kritima
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1. Biotechnology Process

Uploaded by

Kritika Kritima
Copyright
© © All Rights Reserved
Available Formats
Download as PDF, TXT or read online on Scribd
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1.

BIOTECHNOLOGY: Process
13 March 2024 11:28 AM

Definition of Biotechnology:
✓ According to EFB "Biotechnology is the integrated use of biochemistry, microbiology and engineering
sciences in order to achieve technological (industrial) application of the capabilities of microorganisms,
cultured tissues cells and parts thereof."

PRINCIPLES OF BIOTECHNOLOGY
1. Genetic Engineering: Techniques to alter the chemistry of genetic material (DNA and RNA),and to
introduce these into host organisms and thus change the phenotype of the host organism
2. Bioprocess engineering/chemical engineering: Maintenance of sterile (microbial contamination-free)
ambience enables growth of only desired microbe/eukaryotic cell in large quantities for the manufacture of
biotechnological products like antibiotics, vaccines, enzymes etc.

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Two Main Discoveries:
(i) Presence Of plasmids in bacteria- undergo replication along with and independent of chromosomal DNA
(ii) Restriction endonucleases- can break DNA at specific sites. They are appropriately called molecular scis

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TOOLS OF RECOMBINANT DNA TECHNOLOGY
1. Enzymes
2. Cloning Vectors (Vehicle DNA)
3. Competent host (for transformation with recombinant DNA)

1. Enzymes
A. Lysing Enzymes: These enzymes are used to break the cell wall to get DNA.

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B. DNA Manipulating Enzymes: These enzymes are used to break, bind and polymerase DNA molecules.
i. Restriction Enzymes
ii. DNA Ligases
iii. DNA Polymerases

Restriction Enzymes:
• Two enzymes responsible for restricting the growth of bacteriophage in E. Coli were isolated.
One of these add methyl groups to DNA (methylase), while the other cut DNA. The later was
called Restriction Endonuclease.
• First discovered restriction endonuclease Hind II. There are more than 900 restriction enzymes
isolated from over 230 strains of bacteria
• Restriction enzyme recognises palindromic nucleotide sequence in DNA (4-8 nucleotides).

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Nomenclature of Restriction Enzymes.
- Restriction Enzymes are named for the bacterium from which they have been isolated.
- The first letter used for the enzyme is the first letter of the bacterium's genus name (in italics).
- Then comes the first two letters of its species (also in italics).
- The fourth letter of the name of enzyme is first letter of the strain. It is written in capital.
- The end of the name indicates the order in which the enzyme was isolated. It is written in Roman number.

For example, the enzyme Eco RI was isolated from the bacterium Escherichia coli RY 13.

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○ Working of Restriction Enzyme:
▪ Some RE like Eco RI cut DNA little away from centre of palindrome sites, but between the same two bases on the opposite
strands, leaving single-stranded overhanging stretches called sticky ends or cohesive ends on each strand.
▪ Some RE cut the strand of DNA in the centre of palindrome. Such ends are called blunt ends or flush ends e.g., Sma I.

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▪ Some RE cut the strand of DNA in the centre of palindrome. Such ends are called blunt ends or flush ends e.g., Sma I.

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DNA Ligases:
▪ Forms phosphodiester bonds between adjacent nucleotides.
▪ Most common enzyme used in rDNA technology is T4 DNA Ligase, encoded by phage T4.

DNA Polymerases:
▪ These enzymes synthesize a new strand of DNA complementary to an existing DNA
template in 5' to 3' direction e.g., DNA Polymerase I

2. Cloning Vectors (vehicle DNA or carrier of DNA)


- Vector is a carrier/vehicle that delivers a foreign DNA into the host organism

Essential features of a vector are:


A. Origin of replication (ori): Sequence from where replication starts.

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B. Selectable marker: Helps in identifying non-transformants and transformants (only recombinants). SMs
are genes e.g., ampicillin R, chloramphenicol, tetracycline or kanamycin, etc. Also, the gene lac Z
coding for B galactosidase enzyme may also be used as selection basis.
C. Cloning sites: Recognition sites for restriction enzymes. The gene of interest is inserted at restriction
enzyme site.

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pUC8

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Identification of recombinant cells is achieved by a single step i.e., plating cells onto agar medium
containing ampicillin and X-gal (chromogenic substrate for B-galactosidase enzyme encoded by lac Z).

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2. Bacteriophage Vectors: Viruses that infects bacteria
a. Lambda (λ) phage vector: Double-stranded, linear DNA genome in which the 12 bases at each end are
unpaired but complementary called cos sites (cohesive end sites). The lambda genome remains linear in
the phage head, but within E. coli cells the two cohesive ends join to form a circular molecule necessary
for replication.
b. M13 phage vector: It is filamentous phage which infects, E.coli.

3. Cosmid: It is the combination of cos site of lambda phage and plasmid DNA. ('Cos' = Cohesive end sites)
4. YAC Vector: Yeast artificial chromosome contain telomeric sequence, the centromere and autonomously
replicating sequence from yeast chromosome. It is used to clone DNA fragments of size 1000 kbp.
5. Transposons as Vector: These are unit of DNA which can move from one DNA molecule to another.
6. Vectors for cloning genes in plants:
Ti plasmid of Agrobacterium tumefaciens: Agrobacterium tumefaciens, a pathogen of dicot plants delivers
a piece of DNA known as T-DNA to transform normal plant cells into a tumor and direct these tumor cells
to produce the chemicals required by the pathogen. The tumor inducing (Ti) plasmid of A. tumefaciens has
now been modified into a cloning vector which is no more pathogenic to the plants but is still able to use the
mechanism to deliver genes of our interest.

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7. Viruses used to clone genes in animals:
Retroviruses: These viruses in animals have the ability to transform normal cells into cancerous cells.
Similarly retroviruses have also been disarmed and are now used to deliver desirable genes into animal cells.
e.g Adenovirus, Papillomavirus
8. Shuttle Vector: These vector can replicate in both eukaryotic cell and E.coli i.e., these vectors contain two
types of origin of replication and selectable marker genes, one for eukaryotic cell and another for E.coli e.g.,
YEP ( Yeast Episomal Plasmid) and Modified Ti plasmid.

PROCESSES OF RECOMBINANT DNA TECHNOLOGIES

▪ Recombinant DNA technology involves several steps in specific sequence


1. Isolation of DNA (total cell DNA or plasmid)
2. Fragmentation of DNA by RE
3. Separation and isolation of a desired DNA fragment
4. Amplification of gene of interest using PCR
5. Ligation of the DNA fragment into a vector
6. Transferring the recombinant DNA into the host
7. Culturing the host cells in a nutrient medium at a large scale
8. Extraction of the desired product

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