Biotechnology: Principles 3 Flashcards
steps of rdna technology
i) isolation of the Genetic Material (DNA)
ii)Cutting of DNA at Specific Locations
iii)Amplification of Gene of Interest using PCR
iv)Insertion of Recombinant DNA into the Host
Cell/Organism
v)Obtaining the Foreign Gene Product
vi) Downstream Processing
- Isolation of DNA.
- Fragmentation of DNA by Restriction Endonuclease.
- Agarose gel electrophoresis for isolating desired gene.
- Amplification of gene of interest by PCR.
- Ligation of desired gene fragment into vector and thus forms rDNA.
- Insertion of rDNA into host cells/organism.
- Selection of cells having rDNA (Transformant with Recombinant).
- Obtaining the foreign Gene product.
- Downstream processing.
Isolation of the Genetic Material (DNA)
i) In majority of organisms this is
deoxyribonucleic acid or DNA. In order to cut the DNA with
restriction enzymes, it needs to be in pure form, free from other
macro-molecules.
ii)Since the DNA is enclosed within the membranes, we have to break the cell open to release DNA along with other macromolecules such as RNA, proteins, polysaccharides and also lipids. This can be achieved by treating the bacterial cells/plant or animal tissue with enzymes such as lysozyme (bacteria), cellulase (plant cells), chitinase (fungus).
iii) genes are located on long molecules of DNA
interwined with proteins such as histones. The RNA can be removed by treatment with ribonuclease whereas proteins can be removed by treatment with protease.
iv)Other molecules can be removed by appropriate treatments and purified DNA ultimately precipitates out after the addition
of chilled ethanol. This can be seen as collection of fine threads in the suspension. This can be removed by spooling.
Cutting of DNA at Specific Locations
Restriction enzyme digestions are performed by incubating purified DNA
molecules with the restriction enzyme, at the optimal conditions for that
specific enzyme. Agarose gel electrophoresis is employed to check the
progression of a restriction enzyme digestion. DNA is a negatively charged
molecule, hence it moves towards the positive electrode (anode)
(Figure 9.3). The process is repeated with the vector DNA also.
how is the digested dna added
The joining of DNA involves several processes. After having cut the source DNA as well as the vector DNA with a specific restriction enzyme,
the cut out ‘gene of interest’ from the source DNA and the cut vector with space are mixed and ligase is added. This results in the preparation of
recombinant DNA.
Amplification of Gene of Interest using PCR
PCR stands for Polymerase Chain Reaction. In this reaction, multiple copies of the gene (or DNA) of interest is synthesised in vitro using two
sets of primers (small chemically synthesised oligonucleotides that are
complementary to the regions of DNA) and the enzyme DNA polymerase.
steps of process of pcr
-The enzyme extends the primers using the nucleotides provided in the reaction and the genomic DNA as template. If the process of replication of DNA is repeated many times, the segment of DNA can be amplified to approximately billion times, i.e., 1 billion copies are made.
-Each cycle has three steps: (i) Denaturation(94oC);
(ii) Primer annealing(54oC); and (iii) Extension of primers(72oC)
-Such repeated amplification is achieved by the use of a thermostable DNApolymerase (isolated
from a bacterium, Thermus aquaticus), which
remain active during the high temperature induced denaturation of double stranded DNA. The amplified fragment if desired can now be
used to ligate with a vector for further cloning
Insertion of Recombinant DNA into the Host
Cell/Organism
There are several methods of introducing the ligated DNA into recipient
cells. Recipient cells after making them ‘competent’ to receive, take up
DNA present in its surrounding. So, if a recombinant DNA bearing gene
for resistance to an antibiotic (e.g., ampicillin) is transferred into E. coli
cells, the host cells become transformed into ampicillin-resistant cells. If
we spread the transformed cells on agar plates containing ampicillin, only
transformants will grow, untransformed recipient cells will die. Since, due
to ampicillin resistance gene, one is able to select a transformed cell in the
presence of ampicillin. The ampicillin resistance gene in this case is called
a selectable marker
Obtaining the Foreign Gene Product
When you insert a piece of alien DNA into a cloning vector and transfer it
into a bacterial, plant or animal cell, the alien DNA gets multiplied. In
almost all recombinant technologies, the ultimate aim is to produce a
desirable protein. Hence, there is a need for the recombinant DNA to be
expressed.
The foreign gene gets expressed under appropriate conditions.
The expression of foreign genes in host cells involve understanding many
technical details
After having cloned the gene of interest and having optimised the
conditions to induce the expression of the target protein, one has to
consider producing it on a large scale.
what is desirable protein called as? what is the initial prep bfr large scale production
small scale production of culture
If any protein encoding
gene is expressed in a heterologous host, it is called a recombinant
protein. The cells harbouring cloned genes of interest may be grown
on a small scale in the laboratory. The cultures may be used for
extracting the desired protein and then purifying it by using different
separation techniques.
large scale production method
The cells can also be multiplied in a continuous culture system wherein
the used medium is drained out from one side while fresh medium is
added from the other to maintain the cells in their physiologically most active log/exponential phase. This type of culturing method produces a
larger biomass leading to higher yields of desired protein.
bioreactor
Small volume cultures cannot yield appreciable quantities of products.
To produce in large quantities, the development of bioreactors, where
large volumes (100-1000 litres) of culture can be processed, was required.
Thus, bioreactors can be thought of as vessels in which raw materials are
biologically converted into specific products, individual enzymes, etc.,
using microbial plant, animal or human cells. A bioreactor provides the
optimal conditions for achieving the desired product by providing
optimum growth conditions (temperature, pH, substrate, salts, vitamins,
oxygen).
what are the components of a bio reactor
The most commonly used bioreactors are of stirring type.
A stirred-tank reactor is usually cylindrical or with a curved base to
facilitate the mixing of the reactor contents. The stirrer facilitates even
mixing and oxygen availability throughout the bioreactor.
Alternatively
air can be bubbled through the reactor. This takes place in the stirred-sparger type of bioreactor. The oxigen bubbles increase the transfer surface area
If you look at the figure closely
you will see that the bioreactor has an agitator system, an oxygen delivery
system and a foam control system, a temperature control system, pH
control system and sampling ports so that small volumes of the culture
can be withdrawn periodically.
Downstream Processing
After completion of the biosynthetic stage, the product has to be subjected
through a series of processes before it is ready for marketing as a finished product. The processes include separation and purification, which are
collectively referred to as downstream processing. The product has to be
formulated with suitable preservatives.
Such formulation has to undergo
thorough clinical trials as in case of drugs. Strict quality control testing
for each product is also required. The downstream processing and quality
control testing vary from product to product.
