Insertion of recombinant DNA into the host cell/ organism
The vector DNA (
• e.g., plasmid DNA) and alien (foreign) DNA
carrying gene of interest are cut by the same restriction endonuclease to produce complementary sticky ends. With the help of DNA ligase enzyme, the complementary sticky ends of the two DNAs are joined (annealing) to produce a recombinant (chimaera) DNA (rDNA).
Eukaryotic genes do not function properly when cloned
•
into bacterial cell, because of introns. In such cases, DNA is made from mRNA by reverse transcription or synthesised artificially.
This
• rDNA is inserted into host bacterium by transformation using cold CaCl2 solution. The bacterial cell containing the
desired rDNA is selected using selective antibiotic in the culture medium.
Obtaining the desirable gene product
When recombinant DNA is transferred into a bacterial,
•
plant or animal cell, the foreign DNA is multiplied. The cells having cloned genes of interest can be grown on a small scale in the laboratory. The cultures may be used for
extracting and purifying the desired protein. Bioreactors (Fermenters)
Bioreactor provides the optimal conditions for obtaining the
•
desired product by providing optimum growth conditions such as temperature, pH, substrate, vitamins, oxygen and salts.
The most commonly used bioreactors are of
• stirring type.
A bioreactor (fermenter) has a provision for batch culture or continuous culture. In continuous culture, the culture medium is added and the product is taken out.
A stirred-tank bioreactor
• is usually cylindrical or with a
curved base to facilitate the mixing of the reactor content. The stirrer facilitates even mixing the oxygen availability throughout the bioreactor. Alternatively air can be bubbled through the reactor. The bioreactor has an agitator system, an oxygen delivery system and 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system, a temperature control system, pH control system and sampling ports so that small volumes of the culture can be withdrawn periodically.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 through 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.
WorldMags.net
WorldMags.net
MTBIOLOGY TODAY
|
FEBRUARY ’13 79chAPTer-12 : BIOTechnOLOGY AnD ITs APPLIcATIOns Biotechnology
• is the branch of science dealing with the technological use of biological process. It employs molecular biology and genetics to create improved agricultural products, food, animal feed, industrial materials and medicines.
Research areas of biotechnology
• – Following are
three research areas of biotechnology. Providing the best catalyst in the form of
– improved
organism ; generally a microbe or pure enzyme. Creating optimal conditions through engineering for a –
catalyst to act, and Downstream processing
– technologies to purify the
protein/organic compound.
BIOTechnOLOGIcAL APPLIcATIOns In AGrIcuLTure
There are three options to increase the food production :
•
Agrochemical based agriculture; –
Organic agriculture ; and –
Genetically engineered crop - based agriculture. –
The
• Green Revolution succeeded in increasing the food production but it was not sufficient to feed the growing human population. One solution of this problem is use of genetically modified corps. Genes of plants, bacteria, fungi and animals have been changed by manipulations, therefore, these organisms are called Genetically Modified Organisms (GMO).
GM plants have been useful in many ways. Genetic
•
modification has:
made crops more tolerant to
– abiotic stresses (cold,
drought, salt, heat). reduced reliance
– on chemical pesticides (pest-
resistant crops). helped to reduce
– post harvest losses.
increased efficiency of
– mineral usage by plants (this
prevents early exhaustion of fertility of soil). enhanced
– nutritional value of food, e.g., vitamin ‘A’ enriched rice.
Transgenic plants
Transgenic plants can be obtained by
• combination of
tissue culture and genetic engineering. Transgenic plants have a natural resistance to herbicides and pests.
The vector used to introduce new genes into plant cells is
•
most often a plasmid from the soil bacterium Agrobacterium tumefaciens. This is the Ti plasmid (tumour inducing
plasmid), so called because in nature, it induces tumours in broad leaf plants. For using Ti plasmid as a vector, researchers have eliminated its tumor causing properties while keeping its ability to transfer DNA into plant cells.
Hence, for genetic engineering purposes, Agrobacterium strains are developed in which tumor-forming genes are deleted. These transformed bacteria can still infect plant cells.
The part of Ti plasmid transferred into plant cell DNA, is
•
called the T-DNA. This T-DNA with desired DNA spliced into it, is inserted into the chromosomes of the host plant where it produces copies of itself, by migrating from one chromosomal position to another at random. Such plant cells are then cultured, induced to multiply and differentiate to form plantlets. Transferred into soil, the plantlets grow into mature plants, carrying the foreign gene, expressed throughout the new plant.
Bt Cotton (Insect resistant plant) Soil bacterium
• Bacillus thuringiensis produces proteins that
kill certain insects like lepidopterans (tobacco budworm, armyworm), coleopterans (beetles) and dipterans (flies, mosquitoes). B. thuringiensis forms some protein crystals. These crystals contain a toxic insecticidal protein. The Bt toxin protein exists as inactive
• protoxin but once
an insect ingests the inactive toxin it is converted into an active form of toxin due to the alkaline pH of the alimentary canal which solubilise the crystals. The activated toxin binds to the surface of midgut epithelial cells and create pores that cause cell swelling and lysis and eventually cause death of the insect.
Bt toxin genes were isolated from
• Bacillus thuringiensis
and incorporated into the several crop plants such as cotton.
The choice of genes depends upon the crop and targeted
•
pest, as most Bt toxins are insect-group specific. The toxin is coded by a gene named cry. There are numerous genes. Two cry genes cryIAc and cryIIAb have been incorporated in cotton. This genetically modified crop is called Bt cotton as it contains Bt toxin genes against cotton bollworms. Similarly, cryIAb has been introduced in Bt corn to protect the same from corn borer.
Pest resistant plants A nematode
• Meloidegyne incognitia infests the roots of
tobacco plants and causes a great reduction in yield. A novel strategy was adopted to prevent this infection that was based on the process of RNA interference (RNAi). RNA interference (RNAi) is the phenomenon of inhibiting activity of a gene through production of sense and antisense RNA.
This method involves a
• specific mRNA silencing. The