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Title: BIOTECHNOLOGY PRINCIPLES NOTES
Description: Join me in exploring the foundational principles of biotechnology! In these notes, we'll delve into the fundamental concepts that drive innovation in this dynamic field. From genetic engineering to bioprocessing, I'll guide you through the key techniques and applications that shape modern biotechnology. Whether you're a student diving into biology or simply intrigued by the possibilities of biotech, these notes offer clear explanations and insights to deepen your understanding. Let's unlock the potential of biotechnology together!
Description: Join me in exploring the foundational principles of biotechnology! In these notes, we'll delve into the fundamental concepts that drive innovation in this dynamic field. From genetic engineering to bioprocessing, I'll guide you through the key techniques and applications that shape modern biotechnology. Whether you're a student diving into biology or simply intrigued by the possibilities of biotech, these notes offer clear explanations and insights to deepen your understanding. Let's unlock the potential of biotechnology together!
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Biotechnology: Principles and processes
What is biotechnology?
●
Biotechnology refers to the technology using biology, which has
applications in agriculture, food processing industry, medicine
diagnostics, bioremediation, waste treatment, and energy production
...
Basis of Modern Biotechnology
●
Genetic engineering −
Introduction of foreign genetic material
(DNA/RNA) into the host’s genome and altering its phenotype
●
Aseptic techniques −
Involves maintenance of contaminationfree
ambience in chemical engineering processes for manufacture of
products such as antibiotics, vaccines, etc
...
Genetic Engineering
●
Asexual reproduction preserves the genetic information while sexual
reproduction preserves variations
...
●
Genetic engineering overcomes this limitation
...
●
For getting replicated, the foreign DNA must integrate into the host
DNA sequence having ‘origin of replication’
...
This
process is called
cloning
(the process of formation of multiple identical
copies of DNA)
...
●
Plasmid DNA acts asa
vector
since it is used to transfer the piece of
DNA attached to it to the host
...
●
Plasmid DNA was cut with a specific restriction enzyme (‘molecular
scissors’ − that cut a DNA at specific locations)
...
●
The DNA of interest is hybridised with the plasmid with the help of
DNA ligase to form a
Recombinant DNA
...
coli
, where it
replicates by using the host’s replicating machinery
...
coli
is cultured in a medium containing antibiotic, only cells
containing recombinant DNA will be able to survive due to antibiotic
resistance genes and one will be able to isolate the recombinants
...
●
Nucleases are of two types:
○
Endonucleases − Cut the DNA at specific positions within the
DNA
○
Exonucleases − Cut the DNA at the ends (Remove the
nucleotides at the ends of the DNA)
●
Every restriction enzyme identifies different sequences (Recognition
sequences)
...
●
Recognition sequences are
pallindromic
Pallindromes are the
sequence of base pairs that read same both backwards and forwards
(i
...
, same
and
direction)
...
●
As a result, overhangs (called sticky ends) are generated on each
strand
...
●
All these processes form the basis of RDT
...
g
...
coli
, strain R
...
Gel Electrophoresis
●
The fragments obtained after cutting with restriction enzymes are
separated by using gel electrophoresis
...
●
Fragments separate according to their size by the sieving properties of
agarose gel
...
●
Staining dyes such as ethidium bromide followed by exposure to UV
radiations are used to visualise the DNA fragments
...
●
These bands are cut from the agarose gel and extracted from the gel
piece (elution)
...
Cloning vectors & host as tools of RDT
Cloning Vectors
●
Plasmids and bacteriophages are commonly used as cloning vectors
...
●
Bacteriophages − Have high copy number (of genome) within the
bacterial cell
●
Plasmids − May have 1 − 2 copy number to 15 − 100 copy number
per cell
●
If foreign DNA is linked to these vectors, then it is multiplied to the
number equal to the copy number of vector
...
Components of a plasmid cloning vector
●
Origin of replication (
ori
)
○
Replication starts from
ori
...
○
With the help of this, the genetic engineer may control copy
number of the recombinant DNA
...
●
Selectable marker
○
These genes help to select recombinants over nonrecombinants
...
●
Cloning sites
○
These sites refer to the recognition sites for restriction enzymes
(such as EcoRI, Hind III, PvuI , BamHI, etc
...
○
Cloning process becomes completed when more than one
recognition sites are present
...
How antibiotic resistance genes help in selecting recombinants?
●
R
Suppose tet
gene has Bam HI recognition site
...
●
Hence, tetracycline resistance is not present in the recombinants
...
●
On the other hand, nonrecombinants will grow on medium containing
ampicillin as well as on medium containing tetracycline
...
Alternate selectable marker
●
Other than antibiotic resistance genes, alternative markers can be
used
...
●
When foreign gene is inserted within
galactosidase gene, the
enzyme
galactosidase gets inactivated (insertional inactivation)
...
●
Nonrecombinants will produce bluecoloured colonies
...
Cloning vectors for plants and animals
●
Ti plasmid (tumourinducing plasmid) refers to the plasmid of
Agrobacterium tumefaciens
...
tumefaciens
is a plant pathogen
...
○
Ti plasmid can be modified into a cloning vector by removing the
genes responsible for pathogenicity
...
They produce
cancers in animals
...
Competent host
●
Competent host refers to the bacterial cells that have the ability to
take up the vector (containing Recombinant DNA)
...
g
...
Then, these
cells are incubated with recombinant DNA on ice, followed by
heat shock (at 42º), and then putting them back on ice
...
○
Microinjection
− Recombinant DNA is directly injected into the
nucleus of animal cell
...
○
Disarmed vector as in case of
A
...
●
First of all, cells are treated with specific chemicals to break open the
cell to release cellular components such as DNA, RNA, proteins, etc
...
●
Contaminants such as RNA and proteins are digested with the help of
ribonucleases and proteases respectively
...
Cutting of DNA at Specific Locations
●
DNA is cut into fragments with the help of restriction enzymes
...
●
Recombinant DNA is obtained by hybridising ‘gene of interest’ with
vector, with the help of enzyme DNA ligase
...
Several identical copies of
it can be synthesised in vitro
...
●
PCR consists of 3 steps:
○
Denaturation
− Double helical DNA is denatured by providing
high temperature
...
○
Extension
− Replication of DNA occurs in vitro
...
Insertion of Recombinant DNA into Competent Cells
●
Insertion of recombinant DNA into host is done by several methods:
○
Transformation in case of bacteria
○
Disarmed vectors, biolistics, and microinjections in case of plant
and animal cells
○
The cells bearing recombinant DNA are selected because the
recombinants exclusively have selectable marker present in
them (similar to antibiotic resistance)
...
●
The cell containing recombinant DNA will produce a novel protein
product (desirable product/Recombinant protein)
...
●
Continuous culture − Used culture media is drained from one side and
fresh culture media is added from the other side
...
○
Larger biomass is produced by this method leading to higher
yield
...
○
●
○
A bioreactor has the following components agitator system,
oxygen delivery system, foam control system, temperature and
pH control system, sampling ports
...
●
The products so obtained are crude and require separation,
purification, and finishing, which is done under downstream processing
(DSP)
...
●
After proper separation and purification, preservatives are added and
the finished product is made to undergo clinical trials and quality
checks before being sent to market
Title: BIOTECHNOLOGY PRINCIPLES NOTES
Description: Join me in exploring the foundational principles of biotechnology! In these notes, we'll delve into the fundamental concepts that drive innovation in this dynamic field. From genetic engineering to bioprocessing, I'll guide you through the key techniques and applications that shape modern biotechnology. Whether you're a student diving into biology or simply intrigued by the possibilities of biotech, these notes offer clear explanations and insights to deepen your understanding. Let's unlock the potential of biotechnology together!
Description: Join me in exploring the foundational principles of biotechnology! In these notes, we'll delve into the fundamental concepts that drive innovation in this dynamic field. From genetic engineering to bioprocessing, I'll guide you through the key techniques and applications that shape modern biotechnology. Whether you're a student diving into biology or simply intrigued by the possibilities of biotech, these notes offer clear explanations and insights to deepen your understanding. Let's unlock the potential of biotechnology together!