APPLIED BIOTECHNOLOGY

MEDICAL BIOTECHNOLOGY
DISEASE DIAGNOSIS - Biotechnological methods involving probes and monoclonal antibodies have replaced the conventional methods of diagnosis which are tedious and time consuming. - Probes are small nucleotide (DNA/RNA) sequences used to detect the presence of complementary sequences in nucleic acid samples. Probes are bein g used in clinical diagnosis for the detection of microorganisms in various samples. - Monoclonal antibodies are derived from a single cell clone and are specific for a single antigen.
DETECTION OF INFECTIOUS DISEASES

Infectious diseases like Hepatitis C, Leishmaniasis and conditions like AIDS can be detected by ELISA. Kala Azar is detected by a biotechnological method called immuno dot. Western blotting is a confirmatory test of AIDS Human beings suffer from several hundred genetic diseases almost all of which are produced by single recessive mutations. Many of these ailments can be managed but there is no cure for any of them, except for the fast emerging option of gene therapy. Their incidence can be minimized by an early detection of the affected fetuses, which are then aborted. Therefore when a woman gets pregnant the probability of her having a child from a genetic disorder is estimated based on the histories of her and her husbands families and from the knowledge of previous births, if any. In case of risk, further investigation is carried out for a better diagnosis. Diseases like Thalasemia, Haemophilia etc are genetic diseases.

DETECTION OF GENETIC DISEASES

DISEASE TREATMENT (THERAPEUTICS)

USE OF ANTISENSE OLIGONUCLEOTIDES

A very effective and specific approach for treatment of variety of diseases is to design and use oligonucleotides (say 25-35 bases long) complementary to the 5 ends of the parasite mRNAs; such oligonucleotides are called antisense oligonucleotides. The antisense olignonucleotide may be linked to an acridine for increased effectiveness. When such oligonucleotides were used on cultured blood parasite Trypanosoma brycei the parasite was killed. This approach has been quite successful in the treatment of cancer. Due to its enormous potential siRNAs generated by the activity of Dicer and the ssRNA produced from siRNAs by the activity of RISC (RNA induced silencing complex) are being actively considered as an effective tool for the therapeutic inactivation of various pathogenic genes.

RNAi AS THERAPEUTICS

INSULIN

The peptide hormone i.e insulin is secreted by the Islets of Langerhans of pancreas which catabolizes glucose in blood. Mature Insulin consists of two polypeptide chains, A and B linked by disulphide bridges. Its immediate precursor is pro insulin that contains an additional third polypeptide sequence C chain. Insulin is produced initially as pre-pro insulin with A,B,C chains and a small signal peptide linked to B chain. Initial biotechnological attempt was to directly clone the insulin producing gene in microorganisms. But this resulted in an insulin with unwanted C and signal peptides also Later DNA sequences responsible for A and B chains were synthesized separately and cloned separately in two bacteria The chains A and B were isolated form the bacterial cultures and mixed to obtain mature insulin. Recombinant insulin (humulin) is the first therapeutic product produced by means of recombinant technology by Eli Lilly and Company.

SOMATOSTATIN ( GHIH Growth hormone inhibiting hormone)

somatostatin is a peptide hormone produced by the hypothalamus that inhibits the release of growth hormone from the pituitary, and also the release of insulin and glucagons from the pancreas of fasted animals. Somatostatin has two active forms produced by alternate cleavage of a single preprotein; one of 14 amino acids and other of 28 amino acids. Somatostatin was the first human protein obtained from bacteria. Somatostatin is a potentially valuable compound in medicial treatment. Somatostatin gene could not be isolated from human cells so it was synthesized artificially and introduced into E.coli cells through pBR 322 vector. The somatostating gene was inserted at the end of the bacterial gene that encodes for the enzyme beta galactosidase. Once the plasmid was transferred into E.coli, somatostatin was formed as a part of the fusion protein somatostatin-beta glactosidase. By adding cyanogens bromide (a chemical that cleaves proteins at sites carrying amino acid methionine), somatostatin was separated from beta galactosidase. Interferons are groups of proteins called cytokines which are produced by animal and human cells in response to infection by viruses or other agents. They act to inhibit viral replication within the cell. Some also have antibacterial and anticancer properties. They are produced commercially for various therapeutic applications, using recombinant DNA technology. Interferons are produced not only against living virus particles but also against heat killed particles and inactivated virus particles. Once interferons are produced in the body of an animal, the animal maintains the immunity against the viruses for protecting itself during the second attack. Interferons were discovered by Isaacs and Lindemann. Interferons were produced by genetic engineering. The mRNA encoding for interferons were isolated and from it cDNA was prepared. It was transferred to E.coli bacteria through plasmids. The E.coli were cultured and interferons were isolated from the culture Antibiotics are metabolites having antimicrobial activity. Therefore they ae widely used for curing of human diseases caused by microorganisms. Antibiotics are produced by both fungi and bacteria. Among the antibiotics discovered so far, there are four major groups which are most extensively used throughout the world Penicillins, Cephalosporins, Tetracyclines and Erythromycins. Antibiotic producing microorganisms can be cultured for mass production of antibiotics. Alternately the antibiotic producing gene can be cloned in bacteria and their products isolated.

INTERFERONS

ANTIBIOTICS

VACCINES

Conventional vaccines consist of whole pathogenic organism which may be killed or attenuated live microorganisms. They are used to confer immunity against that particular pathogen. This is called vaccination. The collection of enough viruses for vaccination is difficult. So recombinant DNA technology is used to raise new organisms. The vaccines based on recombinant proteins are called subunit vaccines. A portion of the DNA of the disease causing organism is isolated and inserted into E.coli cells. The transferred E.coli cells, receive the pathogenic property and are used as diagnostic agents and as vaccines. Recombinant polypeptide vaccines- Generally the whole protein molecule is not necessary for immunogenicity; the immunogenic property is usually confined to a small portion of the protein molecule. In some cases, the immunogenic protein may be composed of two or more distinct polypeptides. In such cases, it may be desirable to use only one of the polypeptides as vaccine. For example the Cholera enterotoxin (by Vibrio cholerae) consists of three polypeptides A1, A2 and B. The A polypeptides are toxic, while the B polypeptide is non toxic but immunogenic. The gene encoding B polypeptide has been cloned and the recombinant B polypeptide thus produced is being used as a vaccine against cholera. DNA vaccines The gene encoding the relevant immunogenic protein is isolated, cloned and then integrated into a suitable expression vector. This preparation is introduced into the individual to be immunized. The gene is ultimately expressed in the vaccinated individual and the immunogenic protein is produced to invoke the immune system. Biopolymers are polymers produced by living organisms. A hume variety of biopolymers, such as polysaccharides, polyesters and polyamides are produced by microorganisms. These products range from viscous solutions to plastics. The genetic manipulation of microorganisms has permitted the biotechnological production of biopolymers with properties suitable for medical applications such as tissue engineering and drug delivery.

BIOPOLYMERS

DESIGNER DRUGS

Designer drugs specifically and selectively fit into the critical sites of the target molecules, thereby inactivating them. - The target molecule may be an enzyme (concerned with either metabolism or DNA replication), a hormone receptor or some other important molecule involved in a disease. - The aim of drug designing is to develop highly efficient drugs, which have little or no side effects. - There are three main steps in drug designing 1. detailed knowledge (3-D structure) of the critical sites of target molecules 2. designing of drug dmolecules, which will specifically fit into and bind to these critical sites 3. Evaluation of the interaction of the synthesized drugs with the target molecules.

GENE THERAPY

Gene therapy is defined as the introduction of a normal functional gene into cells, which contain the defective allele of the concerned gene with the objective of correcting a genetic disorder or an acquired disorder. Gene therapy is of two types Germline gene therapy and somatic cell gene therapy. In germline gene therapy, germ cells (sperms, eggs or zygotes) are modified by the introduction of functional genes. The change due to therapy is heritable and passed on to later generations. In somatic cell gene therapy the gene is introduced only in somatic cells, especially of those tissues in which expression of the concerned gene is critical for health. It is not heritable. It is of two types augmentation therapy and targeted gene transfer. In Augmented gene therapy an extra gene is inserted but in targeted gene transfer (gene replacement therapy), the defective gene is replaced with a functional one. Gene therapy done at embryonic stages is more successful than in mature stage. Stem cells have the remarkable potential to develop into many different cell types in the body during early life and growth. In addition, in many tissues they serve as a sort of internal repair system. When a stem cell divides, each new cell has the potential either to remain a stem cell or become another type of cell with a more specialized function such as muscle cell, RBC, or a brain cell. Under certain physiologic or experimental conditions, they can be induced to become tissue or organ specific cells with special functions. Stem cells are of two types-embryonic stem cells and non embryonic somatic or adult stem cells. Scientists are using stem cells in the laboratory to screen new drugs and to develop model systems to study normal growth and identify the cause of birth defects. Stem cells have contributed to our knowledge of how an organism develops from a single cell. Human stem cells could also be used to test new drugs. For example, new medications could be tested for safety on differentiated cells generated from human pluripotent cell lines. Stem cells are also used in the generation of cells and tissues that could be used for cell based therapies.

STEM CELLS AND THEIR RELEVANCE

AGRICULTURAL BIOTECHNOLOGY
APPLICATIONS OF PLANT TISSUE CULTURE 1. MICROPROPAGATION - Rapid production of a large number of identical clones within a short duration in the available small space. 2. ELIMINATION OF PATHOGENS - Virus free plants can be produced by meristem culture.

3. GERMPLASM STORAGE - It refers to the collection and storage of different plant species in a limited area. Most of the plants are stored in the form of seeds or as growing plants. Some seeds fail to develop into plants. So identical clones are raised by using tissue culture technique. 4. SOMACLONAL VARIATIONS - Generally clones released through tissue culture methods show uniformity in their characters. But sometimes a few clones show variations among the population of clones found in the culture. These variant clones express new characthers which are not originally found in their parent cells. 5. EMBRYO RESCUE In some plants, normal fertilization takes place, but the ovule fails to become a seed. In such cases, the fertilized egg or immature embryo is removed from the immature fruits and cultured in tissue cultured medium. The resulting individuals are hybrids which show a few new characters. This is called embryo rescue. 6. PRODUCTION OF HAPLOIDS - The haploid plants can be produced by the tissue culture of anther or pollen. These plants are called androgenic haploids. APPLICATION OF TRANSGENIC PLANTS
Bt COTTON

It is a transgenic cotton plant resistant to cotton bollworms (caterpillars) which cause heavy damage to cotton plants. The resistance is conferred by introducing the bt toxin ( proteins) producing gene from Bacillus thuringiensis bacteria to the cotton plant. The transfer of Bt toxin gene into cotton plant is mediated through Ti plasmid vector of Agrobacterium tumifaciens The Bt toxin is a crystal (Cry) protein which kills the bollworms by lysing the gut epithelial cells. It was developed at Swiss Federal Institute of Technology It is a biofortified (nutrient enriched) pro Vitamin A (Beta carotene) rich rice in which the grains are golden yellow in colour The beta carotene gene was isolated from Daffodil plant and a bacteria(Erwinin uredovora) and introduced into rice plants. Golden rice will prevent child blindness caused due to deficiency of Vitamin A

GOLDEN RICE

BIOSAFETY CONCERN - The recombinant DNA techniques initially raised concerns in the scientific community and remain a public concern even today. - It is feared that genetically engineered microorgansisms may disturb the ecosystem in the following ways. 1. They may rapidly multiply and outcompete the native microbes 2. They may also transfer genes related to virulence or pathogenesis into the native bacterial populations and thereby increase their virulence. similarly genetically modified plants could pose biological and ecological risks as follows 1. production of toxic or allergic metabolites

2. unexpected new susceptibilies to pathogens may endanger the ecosystem. 3. The new characteristics may be transmitted to related sexually compatible weed species and to microbial populations 4. ecosystems may be disturbed by dispersal, persistence or altered reaction to parasites, symbionts or competitors. The risk of GMOs should be assessed and biosafety guidelines must be laid for safe use of GM organisms.

ENVIRONMENTAL BIOTECHNOLOGY BIOTECHNOLOGY IN BIODIVERSITY AND CONSERVATION - Biotechnology is defined as any technological application that uses biological systems, living organisms or derivatives thereof to make or modify products or processes for specific use. - Thus the chief and critical raw material for biotechnology is biodiversity. - The relationship between biotechnology and biodiversity is multidirectional 1. Biotechnology provides very powerful tools for the critical assessment of biodiversity-genetics, species and ecosystems and consequently the identification of potential bioresources 2. It gives newer methods and guidelines for the conservation of biodiversity 3. It provides new methods for collecting and storing genes (as seed and tissue culture) 4. Detection and elimination of diseases in gene bank collections 5. Improved techniques for long term storage like cryopreservation. WASTE MANAGEMENT - Human activities have increased the amount of wastes and pollutants that have existed. They have also contributed novel types of wastes like plastic. - The aim of biotechnology should be to develop such processes and products, which minimize the damage to the environment and at the same time are compatible with a high quality of life. - The various approaches to waste treatment are- 1. biofilters (gases) - 2. landfills - 3. incineration - 4. aerobic digestion - 5. anaerobic digestion - The sewage and waste water are properly treated by employing microorganisms. They oxidize the organic pollutants. - Solid wastes (biodegradable) can be converted into compost by the activity of anaeorobic microorganisms. - Genetic engineering methods are adopted for creating new strains of microbes to treat wastes. BIOREMEDIATION

Use of biological organisms such as bacteria, fungi and plants to reduce or eliminate toxic pollutants from contaminated sites by degradation, assimilation or transpiration in the atmosphere is called bioremediation Bioremediation of organic contaminants is primarily based on either microorganisms naturally present at the sites, or on microbial inoculants developed in the laboratory and introduced at the site. Phytoremediation is the use of green plants and their associated microorganisms to remove environmental contaminants. Use of microorganisms like Pseudomonas putida (superbug) to clean oil spills is an example.

INDUSTRIAL MICROBIOLOGY LARGE SCALE PRODUCTION OF BEVERAGES - Enzyme biotechnology plays a very important role in the production of beverages (drinks) both alcoholic and non alcoholic. - Enzymes like polygalacturonase, pectin esterase, pectin lyase and hemicellulases are used for the production of fruit juices and wines. - Enzymes like proteases, fungal alpha and beta amylases, cellulases, papain etc are used for the preparation of beer. - Heat stable bacterial alpha amylases have been used for the production of distilled alcoholic drinks. - The substrate and enzymes are put inlarge fermenters and the product isolated on large scale. LARGE SCALE PRODUCTION OF PHARMACEUTICALS - Enzyme biotechnology also has helped in the large scale production of pharmaceuticals - Penicillin G and V acylase, glucose isomerase etc are widely used in pharmaceuticals for the production of semisynthetic penicillins and fructose syrups. - Among penicillin G acylase producing microorganisms, E.coli strains are most explored and exploited ones. The biosynthesis of penicillin acylase in E.coli is controlled by altering the nutrients and culture conditions. - Penicillin V acylase occurs in fungal and actinomyces sources and some other bacteria.