What are Biotechnology Principles and Processes?
It is defined as the technology that utilizes biological systems, living organisms or their parts to produce commercially or medically important products. ‘The integration of natural science and organisms, cells, their parts and molecular analogues for products and services’.
Principles of Biotechnology
Construction of an Artificial Recombinant DNA Molecule
The gene of interest is identified and is cut at specific locations by the help of restriction enzymes (Molecular scissors).
DNA of interest is attached with the origin of replication which will initiate the process of replication in the host cell.
The gene of interest is linked with plasmid (Extra chromosomal genetic material) with the help of ligase. Plasmid provides antibiotic resistance gene that will help in the process of selection of recombinants.
These “recombinant DNA” (rDNA) molecules are then introduced into host cells, where they can be propagated and multiplied.
Tools of Recombinant DNA Technology
Restriction Enzymes (Molecular Scissors)
Restriction enzymes belong to a large class of enzymes called Nucleases. They are named so because they restrict the growth of bacteriophage in Escherichia coli.
Types of Restriction Enzymes
Nomenclature Of Enzymes
Scientific name should consists of two letters. First should be genus and second should be the name of species of the prokaryotic cell from which they were isolated, e.g., EcoRI comes from Escherichia coli RY 13. Letter ‘R’ is derived from the name of strain. Roman numbers indicates the order in which the enzymes were isolated from that strain of bacteria.
Separation And Isolation Of Dna Fragments (Dna Of Interest)
Fragments of DNA obtained after the action of restriction enzymes are separated by Gel Electrophoresis.
The DNA fragments are separated according to their size. Heavy molecules move slowly and thus can be seen near the walls.
The separated DNA fragments can be visualized only after staining the DNA with Ethidium bromide.
Gel is exposed to UV rays which shows bright orange coloured bands.
The separated bands of DNA are cut out from the agarose gel and are extracted from the gel. This step is known as Elution.
These DNA fragments are purified and used for further experiments.
Cloning Vectors (Vehicles For Cloning)
Vector is any vehicle (Virus, plasmid, bacteria) that help to carry a foreign gene of interest into a given host cell.
Salient Features Of A Vector
Cloning vectors should have following features:
Transformation Of Host Cell
Host cell is made competent to accept the DNA molecule which is hydrophilic in nature.
Cells are incubated with divalent calcium ion Ca2+ to increase the efficiency of cell wall (through pores of cell wall) to take up the rDNA.
rDNA is forced to enter into the cell by incubating both on ice
Then place them briefly at 42°C - Heat Shock
Put them back on ice
Direct Methods of Injecting rDNA Into The Host Cells
Processes Of Recombinant Dna Technology
Sequential steps of rDNA technology are as follows:
Obtaining The Foreign Gene Product
The cells harboring 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.
A bioreactor provides the optimal conditions for achieving the desired product in large quantities by providing optimum growth conditions (temperature, pH, substrate, salts, vitamins, oxygen). There are two types of bioreactors :
(i) Simple Stirred-tank Bioreactor
(ii) Sparged Stirred-tank Bioreactor