Application of Biotechnology

Application of Biotechnology

Biotechnology is the application of scientific and engineering principles to the processing of materials by biological agents to produce goods and services (Coleman, 1986).

Biotechnology may be defined as a technology based on biological systems plants, animals and microbs or parts of it (cell, tissue, genes or DNA) to derive the best goods and services for the benefit of human beings (US. National Science Foundation)

Application of Biotechnology in the field of Agriculture

Insect resistant plant:

In the early part of this century, a scientists named G.S. Berliner found that the sporulating cells of a Bacillus sp. were inhibitory the moth larvae. Berliner named the organisms Bacillus thuringiensis. B. thuringiensis produces toxic crystal in order to cells during the process of sporulation. The toxic substance as an alkaline protein, is deposited on leaves and ingested by caterpillar gut, moth and other related insects. In the caterpillar gut, the insecticide dissolves and the larvae experience paralysis and dead.

            Bacillus thuringiensis (Bt) appears to be harmless to plants and other animals. It is produced by harvesting bacteria at the onset of sporulation and drying them into a commercially available dusting powder. The product is useful on tomato, horn worms and gypsy moth caterpillars. Its success led to discovery of a new strain called B. thuringiensis israelensis (Bti), first isolated in Israel.

A particular gene is responsible for producing toxic crystal protein. Biotechnologist were extracted toxin producing gene (Bt) from B. thuringiensis and the gene to be inserted in the T-region of the Ti plasmid for making recombinant DNA, which is then introduced in to Agrobacterium cells. Such Agrobacterium cells are cocultured with the plant cells into which the gene is to be transferred. The T-region of the Ti-plasmid along with its DNA insert is ultimately integrated into the plant genome. The plant produces a crystal protein which is toxic to most lepidopteran, many coleopteran and other related insects. A truncated version of the (Bt) gene has been transferred into tobacco, tomato, cotton etc.

In 1941, Bacillus popilliae was introduced as a control measure for Japanese beetles. The bacillus infects beetle larvae and causes milky spore disease. So named because the blood of the larvae becomes milky white. The larvae eventually die and the bacillus spore remain in the soil to infect other larvae.

In 1985, Scientists at the Monsanto Company announced the development of a new microbial insecticide for corn plants. Toxic producing genes were extracted from B. thuringiensis and inserted to Pseudomonas fluorescens, a common Gram negative rod that colonizes the roots of corn plants. The Monosanto groups sprayed corn plants with reengineered Pseudomonas and determined that the bacteria would take up residence with the plants, thereby providing a built-in insecticide.

Viruses also show promise as pest control device partly because they are more selective in their activity than bacteria. Once released in the field, the viruses spread naturally. It is also possible to harvest early disease victims, grind them up, and use them disseminate the virus to new locations. Among the insects successfully controlled with viruses are the cotton boll worm, cabbage looper and alfalfa caterpillar.

Herbicides resistance plants:

Herbicides are used in agriculture for killing the weeds (unwanted plants). Normally, herbicide kills both weeds and useful plants by inhibiting an enzyme necessary for making certain essential amino acids. EPSP is an enzyme and play an important role for the production of essential amino acid in plants. Herbicide glyphosate inhibits the shikimate pathway in plants. The same pathway is found in the bacterium Salmonella typhimorium. Glyphosate is an inhibitor of the enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase, a key enzyme of the shikimate pathway. It therefore, blocks the production of chorismic acid which is essential precursor for the synthesis of the aromatic amino acid and essential aromatic metabolites such as artho-amino benzoate. The drawback to glyphosate is that it is equally effective on valuable crops and on weeds.

Glyphosate resistant mutants of S. typhimorium can be isolated by standard mutagenesis. 

Salmonella bacteria happen to have this enzyme and some Salmonella have a mutant enzyme that is resistant to the herbicides. In S. typhimorium, aroA gene is responsible for the production of EPSP enzyme. When the aroA gene for this enzyme is introduced into a croup plants using Ti plasmid co-integrate vector and the leaf disc transformation technique, the croup becomes resistant to the herbicide by producing more EPSP enzyme. The aroA/resistant gene for EPSPS was transferred to Tobacco plants and transgenic tobacco was developed which was resistant to glyphosate herbicide.

There are now a variety of plants in which different herbicide and pesticide resistant have been engineered. Resistance to drought, viral infection and several other environmental stresses has also been engineered into croup plants.

Nitrogen fixation transgenic plant:

Nitrogen is an essential element in nucleic acids and amino acids. Although it is the most common gas in the atmosphere (about 80% of air), animals can not use nitrogen in its gaseous form, nor can any but a few species of plants. The animals and plants thus require the assistance of microorganisms to trap the nitrogen.

Two general types of microorganisms are involved in nitrogen fixation: free living species and symbiotic species. Free living species include bacteria of the genera is Bacillus, Clostridium, Pseudomonas and Azotobacter, as well as types of Cyanobacteria and certain Yeasts. Generally, the free living species fix nitrogen during their growth cycle.  Symbiotic species of nitrogen fixing microorganisms live in association with plants. This plant known as legumes, include peas, beans, soybeans, alfalfa, peanuts etc. Species of Gram-negative rods known as Rhizobium infect the roots of plants and live within swellings or nodules, in the roots. Rhizobium fixes nitrogen into root nodule and makes nitrogenous compounds (NH₃/NH₄) available to plant, while taking energy rich carbon compounds in return.

In Klebsiella pneumonia, nif gene is responsible for the fixation of atmospheric nitrogen. Biotechnologist were isolated nitrogen fixing gene (nif) from Klebsiella and the gene to be inserted in the T-region of the Ti plasmid for making recombinant DNA, which is then introduced in to Agrobacterium cells. Such Agrobacterium cells are co-cultured with the plant cells into which the gene is to be transferred. The T-region of the Ti-plasmid along with its DNA insert is ultimately integrated into the plant genome. The plant fixes atmospheric nitrogen and converted into ammonia.

Improved strains and new species of nitrogen fixing plants should be developed in order to reduce the use of chemical fertilizers in the field.

Production of Virus resistant Plants:

The method most frequency used for producing virus-resistant plants sprang from the observation that, oftentimes, plants infected by nearly avirulent virus are thereafter resistant to super infection by a related, highly virulent one. Thus, deliberate infection with avirulent virus strain has been used to produce protection in crop plants. This phenomenon is known as cross-protection. The method is not totally safe; however, because the avirulent strains many mutate to produce strains that are significantly pathogenic. Most plant viruses are positive strand RNA viruses covered by coat protein sub units. The coat protein gene (cp) from TMV (Tobacco Mosaic Virus) was inserted into tobacco plants genome via the Agro-bacterium using Ti-plasmid vector through disc transformation technique. Tranagenic plants, whose genomes contain introduced tobacco mosaic virus (TMV) coat protein genes and which continuously synthesize the coat proteins, show resistance to virus infection. Transgenic plants showing significant resistance to TMV alfalfa mosaic virus, cucumber mosaic virus, tobacco streak virus and tobacco rattle virus have already been produced in this manner.

Production of Organic Compounds

Microorganisms are used in industry to produce a variety of organic compounds including acids, growth stimulates and enzymes.

Citric acid is one of the first organic acids to be made in bulk by microorganisms. The organism most widely used in citric acid production is the mold Aspergillus niger. Microbiologists inoculate the mold to a medium of corn meal, molasses, salts and inorganic nitrogen in fermentation tanks. The absence of a Krebs cycle enzyme in the mold prevents the metabolism of citric acid and the citric acid accumulates in the medium.

Citric acid is used in food industry (eg. fruit drinks, confectionery, jams, jellies, preserved fruits, candies, wines), pharmecy (eg. blood transfusion ), cosmetics (eg. lotions, shampoos and hair setting fluids) and industries (eg. electroplating, leather tanning, cleaning of pipes, reactivation of old oil wells).

Out line of citric acid production method

Lactic acid is another important microbial product. Several carbohydrate substances corn starch, potato starch, molasses’s and dairy whey can be used for the production of lactic acid.

Lactic acid is commonly produced by bacterial activity on the whey portion of milk. The whey culture is added to the fermentation tank in a volume equivalent to 5-10% of the total volume to be fermented. Tem. 43⁰C.

Lactobacillus bulgaricus is widely used in the fermentation because it produces only lactic acid from lactose.

Any starch used must first be hydrolyzed to glucose by treatment with acids or enzymes.

Lactic acid is used to preserve foods, finish fabrics, prepare hides for leather and dissolve lacquers.

Calcium lactate used in the treatment of calcium deficiency and Ion lactate used in the treatment of anaemia. Sodium lactate is used as Pasticizer and moisturizer.

Enzyme

Enzyme

(Lactose) C₁₂H₂₂O₁₁ + H₂O                  2 C₆H₁₂O₆                          Lactic acid  (CH₃CHOHCOOH)

Gluconic acid another valuable organic acid is useful in medicine as a carrier for calcium. This acid is produced from carbohydrates by Aspergillus niger and species of the bacterium Gluconobacter cultivated in fermentation tanks.

Calcium gluconate is also added to the feed of laying hens to provide calcium that strengthens the eggshells.

Glutamic acid is an organic acid produced by certain species of Micrococcus, Arthrobacter and Brevibacterium.

Glutamic acid is a valuable food supplement for humans and animals and its sodium salt, monosodium glutamate is utilized in food preparations.

Riboflavin (vitamin B₂) and Cyanocobalamin (vitamin B₁₂) are also products of microbial growth.

Riboflavin is a produced of Ashbya gossypii. Cyanocobalamin is produced by selective species of Pseudomonas, Propionibacterium and Streptomyces.

This vitamin prevents pernicious anemia in humans and is most essential for human growth and is also used in bread, flour, cereal products and animal feeds.

Daily requirement of vitamin B₁₂ is about 0.001 mg.

Enzymes and Other products:

Many fungi and bacteria synthesize and secrete industrially useful enzymes into the surrounding medium. For instance, molds such as Aspergillus, Penicillium, Mucor and Rhizopus secrete enzymes that are useful in the processing of a variety of materials. These enzymes include amylase, invertase, protease and pectinase.

Amylase is produced by the mold Aspergillus oryzae. It is used as a spot remover in laundry presoaks, as an adhesive, and in baking.

Invertase, an enzyme from yeast is mixed flavoring agents and solid sucrose and then covered with chocolate. The enzyme converts some of the sucrose to liquid glucose and fructose, forming soft center of the chocolate.

Proteases are a group of protein digesting enzymes produced by Bacillus subtilis, Aspergillus oryzae and other microorganisms.

Proteases are used as liquid glues, laundry presoaks, meat tenderizers, drain openers, and spot removers.

Proteases are also used for bating hides in leather manufacturing, a process in which organic tissue is removed from the skin to yield a finer texture and grain.

In medical microbiology, doctors use another microbial enzyme, streptokinase, to breakdown blood clots formed during heart attack.

Hyaluronidase is another enzyme that used to facilitate the absorption of fluids injected under the skin.

Gibberellins are a series of plants hormone that promote growth by stimulating cell elongation in the stem. This hormone use to hasten seed germination and flowering. This increases the yield of fruit and in the case of graps, enhances their size.

Gibberellins are produced during the metabolisms of the fungus Gibberella fujikuroi and may be extracted from these organisms for commercial use.

Alcohols and Alcoholic Beverages:

Ethanol (ethyl alcohol) is a common solvent and raw materials used in the laboratory and in the chemical industry. It is produced by yeasts using and fermentable carbohydrate as substrate.

Corn, molasses, sugar beets, potatoes and grapes are some of the raw materials used for alcoholic fermentation.

Following are some of alcohol producing microorganisms:

Bacteria: Clostridium acetobutylicum, Klebsiella pneumoniae, Sarcina ventriculi, Zymomonas mobilis etc.

Yeast: Aspergillus oryzae, Saccharomyces cerevisiae, S. oviformis, S. saki, Rhizopus sp., Trichosporium cutaneum etc.

Yeast, S. cerevisiae are usually used for ethanol production.

C₆H₁₂O₆ (Fermented carbohydrate)………………2 CH₃CH₂OH (Alcohol) + 2 CO₂.

Ethanol is a component of alcoholic beverages, and microorganisms are needed to produce such products as wine and beer. Percentage of alcohol differs in different alcoholic beverages.

Wine: It is mainly an European drink produced from juice of fresh grapes (Vitis vinifera). In ripen grapes, concentration of sugar (glucose and fructose) increases. Grapes juice (27% sugar) is fermented by various strains of S. ellipsoideus into alcohol and also reders the chemical constituents which alters the flavour.

The fortified wines (brandy) are prepared on additional of extra ethanol to wines, when fermentation is over, for raising the concentration to about 20%.        

Beer: The word beer is derived from the Anglo-Saxon baere, meaning barley. Beer is produced after the fermentation of mixture barley malt and starch solution by S. cerevisiae.    

Malt is prepared from barley. Grains are allowed to germinate. After 4-6 days amylase and protease are formed. The germinate grains are gradually heated at about 80⁰ C. Malt is mixed with starch cereals (rice, maize, wheat) to produce grist. Mash is prepared by adding hot water to the grist holding for a period to allow enzymatic conversion and draining off the resultant sweet wart. Wart is filtered and then boiled to inactivate enzyme. Then Hops (Humulus lupulus) are added to the wart, giving in flavour, colour and stability. Finally it is fermented with yeast.

After fermentation sugar is converted into alcohol and also brings about minor chemical changes, for example protein.

Brandy is made from fruit juice.

Rum is produced from molasses.

Whiskey is a product of various malted cereal grains.

Votka, which is made from potato starch.

Application of Biotechnology in the Medical Science

Production of Human Insulin:

Insulin is secreted by the Islets of Langerhans of Pancreas tissues which catabolizes glucose in blood. Insulin is a boon for the diabetes whose normal function for sugar metabolisms generally fails.

Diabetes is a common condition and world wide the number of insulin user is more than 2 million. Traditionally, diabetes has been treated with bovine and porcine insulin which is extracted from the pancreas recovered from the slaughtered animals.. The animal insulin is not identical to human insulin. Hence some diabetes produce antibodies against the insulin they need to keep them alive. Therefore, the insulin is destroyed before the body can use it. Such type of this problem can be solved only by using human insulin produced by genetically engineered bacteria which contain the human “insulin gene”.

Human insulin production is completed by the following steps:

1. The desired DNA/genes are selected. They are plasmid of E. coli bacteria and human gene from pancreas which control insulin production.
2. The plasmid of the bacteria is cut/split at certain specific site by the restriction enzyme to ease insertion of human insulin gene.
3. To isolate and cut out the single gene in human cells which control insulin production. The isolation and cutting of the human gene is accomplished by restriction enzymes.
4. The insulin gene (DNA) is inserted into the splitted part of the plasmid using ligase enzyme. In this way, the recombinant DNA is produced.
5. The recombinant DNA is transferred into the E. coli bacteria. In the nutrient media E. coli reproduces rapidly doubling every half hour under favorable condition and make pure human insulin.

It is estimated that cloned of E. coli are capable of producing about one million molecules of insulin per bacterial cell.

Human insulin (Humulin) is the first therapeutic product produced by recombinant DNA technology by Eli Lilly Company & Co. and marketing in 1980.

Vaccine for Foot and Mouth Disease Virus (FMDV):                       

                                                                                                        

Foot and Mouth Disease (FMD) is a serious disease caused by Aphthovirus. The primary control measure of the disease has been the slaughter of FMDV-infected animals. Vaccines are produced by inactivation of virus grown in bovine tongue epithelium. FMDV contains a single stranded RNA covered in a capsid of four polypeptides, for example, VP1, VP2, VP3 and VP4 where only VP1 and VP3 has a little immunogenic activity. Thus, the gene for VP1/VP3 became a target for cloning. However, the nucleotide sequence encoding for VP1/VP3 was identified on the single stranded RNA genome and therefore, it was necessary first to synthesize a double stranded cDNA of entire genome (approximately 8,000 nucleotides long). This cDNA was then digested with restriction enzymes, and the fragments were cloned on double stranded pBR322 in E. coli. About 1,000 molecules of VP1/VP3 per bacterial cell were synthesized (Kupper et.al. 1981).