BIOTECHNOLOGY AND ITS APPLICATION

› Biotechnology deals with industrial scale production of

biopharmaceuticals and biologicals using genetically modified

microbes, fungi, plants and animals.

› Applications of biotechnology includes: therapeutics,

diagnostics, genetically modified crops for agriculture, processed

food, bioremediation, waste treatment & energy production

Introduction

› To supply best catalyst (microbe/ pure enzyme) in the form of

improved organism with better characteristics

› To create optimal conditions by genetic engineering for a

catalyst to function

› Purification of protein/ organic compound by developing

downstream technologies

Major Research Areas of Biotechnology

› Increase in the food production can be achieved by:

1. Agro- chemical based agriculture

2. Organic culture

3. Genetically engineered crop- based agriculture

› Green Revolution- crop production increased by using improved crop varieties, better crop management practices & use of agrochemicals (fertilizers & pesticides)

› Food supply was tripled but was not enough for growing population

› Agrochemicals were too expensive for farmers to afford

› Increase in yield with existing varieties are not possible with conventional breeding

Biotechnological applications in Agriculture

› Introduction of genetically modified crops was an alternative to obtain maximum yield by understanding genetics of the crop

› Some useful aspects of GM plants in agriculture:

1. Improvement in nutritional quality, Eg.- Vitamin ‘A’ enriched rice

2. Better nitrogen fixation 3. Tolerant to abiotic stress (cold, drought, salt, heat)

4. Reduce reliance on chemical pesticides (pest- resistant crops)

5. Reduce post harvest losses

6. Increase efficiency of mineral usage by plants (prevent exhaustion of fertility of soil)

7. Production of diseased resistant crop

8. Micro propagation for bio- mass energy production

Pest Resistant Plants

› Biotechnology in agriculture- Pest resistant plants

› Development of pest resistant plants- reduce the use of pesticide

› One such approach- introduction of Bt toxin to target plant; eg.- Bt

cotton, Bt corn, rice, tomato, potato & soyabean

› Bt toxin- produced by Bacillus thuringiensis due to presence of Bt

toxin gene

› Bt toxin gene- cloned from the bacteria and expressed in plants to

provide resistance to insects (no need of insecticide)- Bio- pesticide

› Bacillus thuringiensis- Bt toxin (protein) which kills insects like lepidopterans (tobacco budworm, armyworm), coleopterans (beetles) & dipterans (flies, mosquitoes)

› Bt toxin gene expresses itself- protein crystals at particular phase of bacteria- insecticidal protein

› Bt toxin exists as inactive protoxins & on ingestion of it by insect- active form of toxin due to alkaline pH of gut- solubilize the crystals

› Activated toxin binds to surface of mid gut epithelial cells & create pores- swellings & lysis- death of insect

› Bt toxin- isolated from bacteria; coded by gene cry; different types

› Incorporated into crop plant depending on type of crop & target pest

› cryIAc & cryIIAb- cotton bollworms & cryIAb- corn borer

Mechanism of Bt toxin & Bt Cotton

Bt (in inactive form) sprayed on Crops

Eaten by insect

Toxin gets activated by alkaline pH of insect’s gut

Swelling of gut of insect

Death of insect

Pest Resistant plants› Bt toxin is not toxic to all pest or pest become resistant against Bt toxin

› Another approach to develop pest resistant crop against nematode parasite- RNA interference (RNAi)

› Eg. Meloidegyne incognita- roots of tobacco

› Principle of RNAi to develop pest resistance- ‘Silencing translation of specific mRNA’

› RNAi takes place in all eukaryotic organism as a method of cell defense

› Silencing of specific mRNA due to complementary dsRNA (a type of RNA molecule to enhance or inhibit translation of mRNA)- binds to mRNA & prevent translation

› Source of dsRNA/ complementary RNA- Virus (RNA) or Transposons- genetic element which replicate via an RNA intermediate

› Vector used- Agrobacterium- genetically engineered to introduce nematode specific genes (DNA)

› Nematode specific gene- introduced into host plant (transgenic plant)

› DNA introduced will produce both sense & anti- sense RNA in host

› Since sense & anti- sense RNA are complementary to each other- double stranded RNA (dsRNA)- initiates RNAi

› When nematode infest the plant- introduction of nematode genome takes place & presence of dsRNA- silence specific mRNA of nematode- initiation of RNAi

› Parasite will not able to survive in host which express interfering RNA (RNAi)

› Transgenic plant- resistant & protected from parasite

Method to genetically modify crop through RNAi

Meloidegyne incognita

Biotechnological applications in Medicine› Biotechnology in medicines

1. Diagnose & treat different diseases with effective recombinant therapeutic drugs

2. Protect from dangerous diseases

3. Through gene therapy new & healthy genes can be inserted to replace damaged cells

4. Targeted action of drug by thorough study of genomics

› Recombinant therapeutics do not induce unwanted immunological responses as in case of similar products isolated from non- human source

› 30 recombinant therapeutics- approved for human use & 12 of these- marketed in India

› Deficiency of Insulin causes Diabetes which can be controlled by

taking insulin at regular intervals

› Traditionally when human- insulin were not available, insulin of

animals (slaughtered cattle & pigs)

› Animal insulin- trigger some immune response like allergy and some

reaction due to foreign insulin & will not be effective

› Bacteria were genetically engineered- produce human insulin in large

quantities as bacteria can easily grown in culture medium

Genetically Engineered Insulin

› First created 1983, Eli Lilly an American company

› Insulin- hormone to regulate blood sugar level & administered in patients with Diabetes

› Mature insulin- two short polypeptide chains: chain A & chain B which are linked by disulphide bridges

› Insulin- synthesized as pro- hormone in all mammals- A peptide, B peptide & additional C peptide- absent in mature Insulin as it get removed during maturation

› Using rDNA technology two DNA sequences corresponding chain A & B of human insulin- introduced into plasmids of E. coli- produce insulin chains separately

› Chains A & B- extracted & combined by disulphide bonds- human insulin

Genetically Engineered Insulin

Maturation of pro- insulin to Insulin

Genetic Engineering of Insulin

› Is a corrective therapy to correct or treat hereditary diseases (a gene defect) diagnosed in a child/ embryo

› Method where genes- inserted into cells & tissues to treat disease

› Principle of Gene Therapy:

Normal gene when get inserted into individual cell or embryo with gene defect the normal gene will take over the function of and compensate for the non- functional gene

› First clinical gene therapy- 1990 to 4 year old girl with Adenosine deaminase (ADA) deficiency

› Initially treated- bone marrow transplantation & by enzyme replacement therapy

Gene Therapy

› ADA deficiency disorder- hereditary disorder caused due to deletion of gene for adenosine deaminase

› Enzyme Adenosine deaminase- crucial for immune system function

› In gene therapy lymphocytes from blood of patients are isolated & grown in culture medium

› Functional ADA cDNA- introduced into lymphocytes through retroviral vector & returned to patients

› Since lymphocytes have short life span- genetically engineered lymphocytes are periodically introduced into patients

› But if gene isolate from marrow cells producing ADA introduced into cells in embryonic stage- permanent cure

Method to treat ADA through Gene therapy

Gene therapy

› Technique- used to diagnose and monitor disease, detect risk, and decide which therapies will work best for individual patients

› Treatments of a disease be effective- early diagnosis & understanding pathophysiology is very important

› Conventional methods of diagnosis (serum & urine analysis)- early detection of diseases- not possible

› Early diagnosis- possible with advanced molecular diagnostics like recombinant DNA technology, Polymerase chain reaction (PCR), Enzyme Linked Immuno- sorbent Assay (ELISA)

› Conventional methods- detect presence of pathogens (bacteria & virus) when disease is well established & produced disease symptoms- pathogen high body

Molecular Diagnostics

Significance of Molecular Diagnostics:1. Molecular diagnostics like PCR detects pathogens even at very

low concentration- amplification of nucleic acids of pathogens

2. Detect HIV (Human Immuno Deficiency Virus) in suspected AIDS patients

3. Detect mutations in genes in suspected cancer patients

4. Detect many genetic disorders

› PCR requires single stranded DNA or RNA tagged with radioactive molecule to hybridize complementary DNA in clones

› Following hybridization the next step- autoradiography

› Clone with mutation (mutated gene)- not appear in photographic film (probe will not have complementarity with mutated gene)

› ELISA- another molecular diagnostics to detect antigen (foreign substance/ pathogen)

› Works on principle- antigen- antibody interaction

› ELISA- detects pathogen by presence of antigens (proteins, glycoproteins, etc.) or by antibodies synthesized against pathogens

› May be defined as animals that have their DNA manipulated to possess and express a foreign gene are known as transgenic animals

Transgenic Animals

1. Normal physiology and development:› Transgenic animals are designed to study different aspects of genes,

like how genes are regulated, how they affect normal function of body & its development.

› For eg.- Study of Insulin like growth factor- involved in growth can be done by introducing genes from other species that alter the formation of this factor & study the effect that results, also information is also obtained about biological role of factor in body

2. Study of disease: › Transgenic animals- model for human diseases in which

investigations of new treatments for diseases & also gives understanding how genes contribute to development of disease

› Transgenic animal model- cancer, cystic fibrosis, rheumatoid arthritis & Alzheimer’s disease

Reasons to produce Transgenic animals

3. Biological Products:› Products produced by living organism some of which are

medicines- expensive

› Transgenic animals can be created by the introduction of genes which codes for a particular product- induce organism to produce desirable product in medium. Eg.-human protein (α-1-antitrypsin) to treat emphysema

› attempts are being made for treatment of phenylketonuria (PKU) and cystic fibrosis.

› In 1997, the first transgenic cow, Rosie, produced human protein-enriched milk (2.4 grams per litre)- the human alpha-lactalbumin and nutritionally more balanced product for human babies than natural cow-milk

4. Vaccine Safety:› For vaccine safety vaccines are first tested on transgenic mice,

developed for use in testing before they are used on humans.

› Transgenic mice are being used to test the safety of the polio vaccine.

› If successful and found to be reliable, they could replace the use of monkeys to test the safety of batches of the vaccine.

5. Chemical safety testing› Transgenic animals- introduced with genes which make them more

sensitive than non- transgenic

› They are then exposed to toxic substances- effect studied

› Toxicity testing in such animals will allow us to obtain results in less time.

› Manipulation of living organisms by the human race requires certain regulations. Ethical standards are required to evaluate the morality of all human activities that might help or harm living organisms.

› Genetic modification of organisms- have unpredictable results when such organisms are introduced into the ecosystem

› Indian Government has set up organizations such as GEAC (Genetic Engineering Approval Committee), which will make decisions regarding the validity of GM research and the safety of introducing GM-organisms for public services.

› The modification/usage of living organisms for public services (as food and medicine sources, for example) has also created problems with patents

Ethical Issues

› Use of genetic materials, plants & biological resources by companies granted with patent has triggered public anger since they have been identified, developed, & used by farmers & people of specific country

› Over 200,000 varieties of rice- found in India which makes India richest

› Basmati rice one type of rice with unique aroma & flavor and there are 27 documented varieties of Basmati- grown in India

› 1997 an American company got patent rights on Basmati rice through US Patent and Trademark office

› This will allow the company to sell a ‘new’ variety of Basmati, in the US and abroad.

› This ‘new’ variety of Basmati had actually been derived by crossing with semi-dwarf varieties and claimed as an invention or a novelty.

› The patent extends to functional equivalents, implying that other people selling Basmati rice could be restricted by the patent.

› Several attempts have also been made to patent uses, products and processes based on Indian traditional herbal medicines, e.g., turmeric neem

› We should be vigilant and counter these patent applications, else other countries/individuals may encash on our rich legacy and we may not be able to do anything about it.

› The patenting of plants, genes, and other biological products that are indigenous to another country

› Biopiracy- use of bio-resources by multinational companies and other organizations without proper authorization from the countries and people concerned without compensatory payment.

› Developed countries patent the knowledge and resources of underdeveloped countries and enjoy immense profits.

› Industrialized nations are rich financially but poor in biodiversity and traditional knowledge. In contrast the developing & underdeveloped world is rich in biodiversity and traditional knowledge related to bio-resources.

› Traditional knowledge related to bio-resources can be exploited to develop modern applications and can also be used to save time, effort and expenditure during their commercialization.

Biopiracy

› Realization of the injustice, inadequate compensation and benefit

sharing between developed and developing countries led some

nations to develop laws to prevent such unauthorized exploitation of

bio-resources and traditional knowledge.

› The Indian Parliament has cleared the second amendment of the

Indian Patents Bill, that takes such issues into consideration,

including patent terms emergency provisions and research and

development initiative.