Genes expression in prokaryotes

J. Lederberg and E. I. Tatum (1946) demonstrated sexuality in bacteria for the first time and this opened a new era of research. Most of the important work on genetics of bacteria was initially done on the colon Bacillus bacteria named Escherichia coli.
Bacteria genome is represented by a circular double stranded DNA. Its DNA is associated with few proteins. E. coli contains 2000-3000 genes. Bacterium can survive on glucose diet giving the idea that these genes have information for synthesis of all organic compounds it needed.
Modes of genetic transfer in bacteria
Bacteria mainly reproduce asexually by binary fission. Meiosis is lacking. They do not show sexual reproduction like eukaryotes. However, some bacteria show primitive form of sexual reproduction to exchange genetic material between two cells. There are three modes of exchange of genetic materials or genetic recombination. They are:
a) Transformation
b) Transduction
c) Conjugation
a) Transformation – A short fragment of naked DNA isolated from one type of bacteria cell is incorporated into other type of bacterial cell. A recombinant or hybrid DNA is, thus, formed. This phenomenon is called as transformation. In this way, DNA of donor cell expresses some of its properties into the recipient cell. Griffith confirmed it with experiment on Diplococcus pheumonia. Normally E. coli does not pick up foreign DNA but can be done in the presence of calcium chloride.
b) Transduction – This is similar to the transformation but transfer of DNA from one bacterium to another is mediated through a vector. This process is called transduction. A vector may be a bacteriophage (virus) or a plasmid or a cosmid.
During this process, bacteriophage is used as a plasmid to transfer a small piece of double stranded DNA from one bacterial cell (so called donor cell) to another cell (so called recipient cell). A recombinant or hybrid DNA is, thus formed which consists of genome of both bacteria and thus expresses both of the properties.
c) Conjugation – Transfer of DNA from one cell to another through a sexual mating is called conjugation. It is similar to sexual mating in eukaryotes. Lederberg and Tatum first demonstrated it in E. coli.
In this process, male bacterium makes sex pili which enables cell to cell contact. The male bacterium is represented as a donor (F?). The female bacterium (recipient F?) lacks pili and receives the DNA from the male bacterium. This result to new genetic recombination. The progeny of the recipient then expresses some of the donor’s traits due to recombination.
Fertility factor and Hfr strain
The ability of transferring genetic material from male is regulated by sex or fertility factor (F genes) present in a plasmid. The bacteria with F genes are said to be F positive (F?) and another F negative (F?). F genes code for producing sex pili and other functions required for transferring DNA. At times F factor integrates into the bacterial chromosome
Such bacteria can transfer their genetic material into female with high frequency (Hfr) in a particular sequence. The are called as Hfr strains. Frequency of recombination was very low in Lederberg’s experiments. Hayes (1952) found a strain of E. coli in which the frequency of recombination was as high as 100 to 1000 times as reported by Lederberg. The strain was called as high frequency recombinant (Hfr) strain.

Other fermented alcoholic beverages

Fermented alcoholic beverages are consumed all over the world. In some country the use of a particular beverage has been passed down from ancient times, some of the alcoholic beverages is explained here:

1. Wine: wine is the product made by the normal alcoholic fermentation of the juice of ripe grapes (vitis vinifera ). Relatively small enounce of wine are made from apples, raisins, black berries, peaches, cherries, orange, currents, apricots, grape fruit, pomegranates, raspberries, pears, honey and straw berries. The wine made from the fruits is named after the fruits, for example apple wine.

2. Whiskey: Whiskey is an alcoholic distillate from the fermented mash of grains. Whiskey is obtained from a fermented mash. After several distillation of mash the low wines are resulted. Further distillation straight whisky. At first several principles are present, wines makes whiskey harsh and unpalatable. The whiskey is aged in charred oak containers. At first whisky is colourless , the colour develops during the aging process. A continued distillation of high wines results in the fermentation of neutral spinets, which are used in blended whiskies and cordials.

3. Rum: Rum is an alcohol distillation from the fermented juice of sugarcane syrup, sugarcane molasses or other sugarcane byproduct. Rum is manufactured and used in general in those countries which grew sugarcane or export molasses or other sugarcane products. It possesses a characteristic flavor, aroma and colour. The flavor and aroma improve with aging. Rum contains about 41% alcohol. Rum is usually aged in charred white oak barrels. Rum may be used in the perspiration of ice-cream, candies in the curing of tobacco, as a beverage and as medicinal.

4. Brandy: Brandy is distillate form of wine. It is also distillate from the remounted juice of various fruit. The best brandy is made in France known as cognac. The other French brandies are known as annoyance. The finest grades of brandy are made from white wines. The brown colour of brandy is coloured with caramel. It contains about 65 to 70 percent alcohol. Apple brandy is known as apple jack.

5. Gin: It is obtained by distillation from a fermented mash of malt or raw grain. The finest gin is distillated from a malt of barley and rye. It requires several distillation. The flavor of gin and any medicinal value are due to oil of jumper.

Application of fermentation in industries
Beside alcohol, other products are also formed by process of fermentation. The product produced depends upon the nutrient medium. Main products are n-propanol, butanol, phenyl ethanol, amyl alcohol, glycerol, lactic acid, acetic acid, pyruvic acid, succinic acid, ethyl acetate, caporic acid, etc. Yeast is also used as animal feed, yeast extract, food supplements and vitamins. Some of important industrial use of fermentation are:
1. Organic acid
a) Citric acid produced in commercial scale from fermenting molasses or purified glucose syrup from maize using Aspergillus niger as microorganism. This fungus also produces gluconic acid.
b) Lactic acid is produced using thermotolerant or thermophilic lactic acid bacteria.
c) Acetic acid most important acid, is produced by the fermentation of carbohydrates

2. Amino acids
Commercial production of number of amino acids is being done using fermentation technology. Out of 20 naturally occurring amino acids, twelve are produced by industrial fermentation or enzyme conversion.

3. Vitamins
Vitamins can be synthesized by chemical processes or biological means. Riboflavin is produced from the fungus Eremothecium ashbyii where vitamin B12 is chiefly synthesized from Streptomyces griseus. Streptomyces also produces antibodies. During fermentation process, change in the operational conditions may lead to the production of vitamin rather than antibiotics.

4. Enzymes
Several thousand enzymes possessing different substrate specificities are now known. The bulks of enzymes used commercially are obtained from microbial, sources, and are produced by fermentation processes. Enzymes from microbial, rather than plant or animal, sources are usually used because of their availability, grater stability and their variety and case of genetic manipulation. The industrial enzymes are produced from different species of bacteria and fungi.

Fermentation process

Fermentation is done with one of the following method

a) Batch process – in this process, nutrient medium and fermenting organism like yeast are allowed to remain in the bioreactor (fermentation tank) till the maximum fermentation product is produced. The entire medium with organism and the product is removed after each charge. Then the bioreactor is cleaned and a fresh batch is stored.

b) Continuous process – In the process, product is drawn off at regular intervals and fresh medium is introduced into bioreactor

c) Immobilized fermentation process – Living yeast cells are immobilized in calcium alginate beads. The beads with living cells are placed in the nutrient medium in fermentation tank. This technique allows quick fermentation about 20 times faster than the batch process.
CO2 is a bye product of alcohol fermentation. It is collected separately. Yeast cells are also isolated from fermentation product. A part of yeast is kept further inoculation. The remaining part of the yeast is washed, dried and employed as animal feed.

Fermentation process involves two broad groups –
I. Upstream process – All the operations before starting the fermenter are collectively called upstream process such as sterilization of the fermenter, preparation and sterilization of culture medium and the preparation and growth of a suitable inoculums of microbial strain.

II. Downstream process – All the operations after the fermentation are known as downstream process. It includes the purification of fermented products from fermentation broth. Methods commonly used are distillation, centrifugation, filtration, and solvent extaction.

Bioreactor and its type
A bioreactor or fementer is a container designed to provide an optimum environment in which microorganisms or their enzymes can interact with a substrate and form a desired products.

Bioreactors are of two types:-
a) Open type: It allows continuous processing with substance entering at one end and products leaving at the another end.

b) Closed type: In this type, the fermentation is done in batches. Microbes are grown on nutrients placed in vessel at the start of fermentation. The stirred-tank fermenter is a versatile design and is used in a range of sizes from one litre laboratory unit to production-scale vessels of typically 100-ton capacity. Its vessel is cooled by a water jacket. Air is pumped into bottom of the liquid, and acid or alkali added as necessary. A stirrer keeps the contents well mixed. Steam lines are provided so that the vessel can be sterilized after each fermentation batch.

During fermentation it is necessary to regulate many factors within predetermined valves. These O2 and CO2, pH, temperature and media concentration, etc. It is also essential to maintain high degree of sterility within bioreactor. It should be made of stainless steel or copper because such bioreactor is resistant to steam sterilization.

Fermentation Technologies in biology

Fermentation is a process of incomplete oxidation of sugar, especially glucose, into alcohol and CO2. It is generally an anaerobic process. By this process, glucose is splitted up by glycolysis into pyruvic acid which is further degraded enzymatically to alcohol and CO2. Several species of yeast, bacteria and other fungi are essential as they contain many enzymes that are used to carry out fermentation.

Fermentation was discovered by ancient people by prolonged soaking (steeping) of grains before cooking or storing juices of fruits. Alcohol was the first product of biotechnology. Now a day a modern technology developed for the production of alcohol on industrial scale is the fermentation.

Yeast and its types
The organism presently used in alcoholic fermentation is the yeast. It is a microscopic fungus Saccharomyces cerevisae. Louis Pasture in the middle of nineteenth century first reported the role of yeast in fermentation process. Presently, yeast products for human and animal consumption are products in large scale in many countries.

There are basically two types of yeast:-
1. Baker’s yeast – These include the selected strains of Saccharomyces cerevisae and Torulopsis grown on molasses. These are used to:
I. Flavor the food
II. Supplement nutrient ingredient as it is rich in proteins, vitamins, etc and
III. Ferment and rise dough in bread making (leaving agent). Leavening is caused by three enzymes secreted by yeast. These three enzymes are amylase, maltase and zymase. Amylase hydrolyses starch into maltose, maltase degrades maltose into glucose and zymase causes fermentation of glucose in anaerobic and produces mainly ethyl alcohol and CO2. Both CO2 and ethyl alcohol evaporate and make the bread porous and soft.

2. Alcohol (Brewer) yeast – These yeast are grown on carbohydrate, sucrose for the production of several types of alcoholic beverages. Majorities of alcoholic beverages are manufactured by metabolic activity of Saccharomyces cerevisae and other yeast.
How and why do microorganisms make alcohol?

Like other organisms, yeast has property to maintain the stock of ATP, which possible due to the consumption of sugars like glucose and fructose. Sucrose is the main component of sugarcane juice which consists of one glucose molecule attached to one molecule of fructose.

If the yeast is grown in oxygenated medium, the sugar will be broken down step by step, into smaller and smaller molecules and at the end, CO2 and energy are liberated. If yeast is grown in anaerobic medium, a series of chemical breakdown processes can be completed and sugar is broken down into ethanol or fuel alcohol.

Glucose is splitted up into two molecules of pyruvic acids via glycolysis. During fermentation pyruvic acids are degraed enzymatically into alcohol and CO2.

Yeast, which carries out alcoholic fermentation, contain two important enzymes –
(1) Pyruvate decarboxylase (2)Alcohol dehydrogenase. The pyruvate decarboxylase catalyzer decarboxylation of pyruvic acid to acetaldehyde which is further reduced to ethanol by NADH in presence of alcohol dehydrogenase.

Process of alcohol manufacture in industries
The raw material used for preparation of ethyl alcohol is molasses. It is thick, dark syrup drained from raw sugar during the refining process. It is also called the products of sugar (in sugar industry). Molasses mash (mixture of raw sugar and hot water) is adjusted to the desired sugar concentration and temperature by addition of water and to the desired pH by addition of a measured quantity of acid or base. Molasses is kept in a bioreactor (fermentation tank). Yeast (Saccharomyces), so called starter, is added and mixed uniformly with molasses mash in the same bioreactor. The mash and starter become well mixed as they spatter and fall to the bottom of the tank. Fermentation soon becomes vigorous with evolution of large quantities of CO2. Fermentation is completed within fifty hours or less. Then the fermented molasses is distilled and separated the alcohol and other volatile constituents from mash. The purification of alcohol is made by means of rectifying columns and then stored in a container.

Applications of recombinant DNA technology

Recombinant DNA technology has opened up new opportunities for highly specific manipulation of the genetic materials. This technology broadens the possibilities of gene transferring gene between unrelated organisms and creating novel genetic information by specific alteration of cloned genes. Recombinant DNA has wide applications in the fields of agriculture and medicine and human health which are discussed herewith.

A. Application of genetic engineering in the field of medicine and human health
The recent results of recombinant DNA technology have revolutionized the field of clinical medicine. This has greatly developed the preparation of wide range of vaccines, development of highly specific diagnostic laboratory tests and prenatal diagnosis of human genetic diseases. Some of the products synthesized using genetic engineering are given below:

1. Vaccines – vaccine is preparation, which contains an antigen composed of whole disease causing organisms. Vaccines are used to confer immunity against antigens.
Recombinant DNA technology can be used to clone the gene for the protective antigen protein. A number of vaccines against virus have been developed using this technology. These vaccines are – Hepatitis B, influenza, HIV (AIDS), Herpes, Foot and Mouth Disease, etc.

2. Human growth hormones – Various human growth hormones have been synthesized commercially using Recombinant DNA Technology. This hormone is used in treatment of dwarfism.

3. Insulin – Insulin, a small protein hormone, is used for controlling diabetes. Bacterially produced human insulin using recombinant DNA technology is now available in market. Products of microbial origin are often safer than those derived from traditional sources. Insulin, produced by microbes does not cause allergic reaction.

4. Interferons – interferons are the glycoproteins which have an inhibitory action upon the multiplication of viruses in cells more or less adjacent to the affected ones. They are being used for the treatment of several diseases including a rare form of cancer called hairy cell leukemia. Various interferon are formed such as – a – interferon, ß – interferon and ? – interferon. The bulk of a – interferon is produced by buffy coats (leukocyte pellets), ß – interferon by fibroblasts and ? – interferon by B or T lymphocytes. Japanese companies have succeeded in producing cloned ? – interferon. Interferon are being used for the treatment of several diseases.

5. Monoclonal antibodies – Antibodies are specific proteins produced by the immune system in response to presence of a specific antigen. Monoclonal antibodies can be produced using hybridoma technology.

Hybridoma technology is used to fuse a normal antibody producing lymphocytes (B-cells or plasma cells) with myelonema cells (a kind of tumor cells) giving a hybridoma. Hybridoma has the potential to grow indefinitely in culture and hence can be a source of unending supply of antibody of choice. Since antibody produced by a hybridoma is biochemically pure, it is called monoclonal antibody. Monoclonal antibodies are used to develop effective vaccines against human, animal and plant diseases.

6. Antibiotics – Antibiotics are the chemical substances, produced both by microorganisms and synthetically. They can inhibit the growth of bacteria and others microorganisms and even destroy them. Penicillin is the first antibiotic discovered by Alexander Flemming in 1928 using recombinant technology. Some useful antibiotics being produced on the commercial scale employing biotechnology technique.

7. Diagnosis of infection disease – Modern medical practice depends on laboratory tests for the specific and correct diagnosis of many diseases. Recombinant DNA technology allows for the production of highly specific diagnosis tests.

Some of the common infectious diseases are cholera, small pox, measles meningitis, hepatitis, etc. These diseases lead the serious damage to the human health. Infectious diseases diagnosis mainly depends upon isolation and identification of pathogens, which may take several days. Development of diagnostic kits to identify pathogenic organisms by knowing the organism-specific DNA sequence has provided rapid, specific and correct diagnosis.

In this way, advancement in biotechnology has made easy early, correct and quick diagnosis of infectious diseases. Various diagnostic kits have been developed for AIDS, cancer, foot and mouth diseases, tuberculosis, etc. Different biotechnological tools used in diagnosis of infectious diseases biotechnology tools used in diagnosis of infectious diseases and prenatal diseases are ELISA, PCR based technique, RIA Essays, etc.

Application of genetic engineering in the field of agriculture

During the last decade, tremendous progress has been made in the area of genetic engineering towards agriculture. Recombinant DNA technology has opened up new opportunities for highly specific manipulation of the genetic material. Once applied and developed to a sufficient degree, it promises ultimately to provide a powerful additional tool to the plant breeders. This technology broadens the possibilities of transferring genes between unrelated organisms and creating novel genetic information by specific alternation of cloned genes. Application of genetic engineering in field of agriculture is:

1. Creation of resistance varieties of plants – Transfer of specific genes from one species to another may be of great significance in exploiting diseases, insects and pest resistance mechanisms more efficiently. Various genes responsible for resistance to diseases have been identified, cloned and incorporated or manipulated into another species. For example, Bacillus thuringiensis is a bacterium that has ability to destroy stem borer pest in rice and maize. Keeping this view in mind, scientists isolated Bt gene from a bacterium Bacillus thuringiensis and cloned and then incorporated it into rice or maize genome. As a result rice or maize shows the resistance against stem-borer due to presence of Bt gene in rice genome. Various novel plants with resistance to various diseases pests and stresses have been created using recombinant DNA technique.

2. Bio-fertilization – Molecular nitrogen in the atmosphere is converted into biologically usable form by nitrogen fixing micro-organisms e.g. Rhizobium. The most sophisticated approach to bio-fertilization is to create plants that possess genetic capacity for nitrogen fixation. Attempts are being made to transfer genes for nitrogen fixation gene called nif gene from bacteria to rice or other non-leguminous crops.

3. Increase the protein content – One of the major source of protein for human and animals consumption is constituted by the proteins contained in seeds of many plant species. The cereals and legumes which are major sources of storage proteins, contain limited amount of certain amino acids which are essentials for human beings. Majorities of these cereals are deficient in lysine whereas legumes are deficient in sulphur amino acids.

The genes which code for a number of storage proteins have been cloned. For example, the gene encoding the French bean protein, phaseolein, has been expressed in sunflower.

4. Creation of transgenic animals – animals that have foreign genes inserted into their germ lines are called transgenic. They can pass the gene on to their offspring, and it can be inherited as a Mendalian trait.

Possible dangers of genetic engineering
Genetic engineering has numerous potential benefits, some of which have been discussed above. However any new scientific discoveries offer the possibility of both beneficial and destructive effects. Some of the possible dangers due to genetic engineering might be as follows:

1. Due to manipulation of genes might, by accident, result in the origin of various new kinds of diseases or organisms containing fatal genetic element.

2. There is a risk of creation of drug resistance germs and out-break diseases against which there is no known prevention.

3. Any accidental escape of laboratory strains may create havoc on earth. It may contaminate a large population.

4. Introduction of gene like that of viral cancer into bacteria through plasmids may involve the risk of introducing these harmful genes into man when these bacteria infect human.

5. Hybrid genomes may create some serious ecological problems, the nature of which is still unknown. Naturally all these harmful hybrids possibly raise many moral, ethical and legal questions.

Releasing the risk involved in genetic engineering by a group of scientists working on recombinant DNA technology, the US National Institute of Health (NIH) established an advisory committee in 1976. Its main objectives are to evaluate the potential of biological and ecological hazards of recombination DNA molecules and develop the procedures which will minimize hazards and devise guidelines for the investigators in the research line.

Genetic Engineering

Genetic engineering can be defined as the science of adding, removing or replacing genetic units in order to achieve permanent and heritable changes in plants and animals for the benefit of mankind. Terms such as “gene surgery”, “gene therapy”, “gene manipulation”, “gene transplantation”, etc. have been synonymously used with genetic engineering. Basically the term genetic engineering refers to techniques that are used to manipulate, move, recombine and propagate DNA.

A detailed knowledge of the molecular nature of the gene and ability to manipulate cells of higher organisms as well as bacteria may eventually allow the possibility of genetic engineering. It has been very useful in plants and animals and has brought biological revolution. Luciferase gene from firefly has been introduced in tobacco plant enabling scientists to study the activation of genes of genes of their choice by measuring the light emitted by the plants. Introduction of nitrogen fixation gene (NIF gene) and Bt gene in cereals are really a boon in agriculture. In human being, genetic engineering is being used for diagnosis of hereditary diseases and thus has bright prospects to be used for gene therapy.

Genetic engineering provides great promises for the improvement of crop plant. Genetically engineered crops with better nutritional status, resistance to insects, pets and herbicides, resistance to fungal, bacterial and viral diseases and resistance to environmental stresses have shown a great potential. The techniques of biotechnology have already increased the capacity to enhance plant productivity. Similarly it has tremendous impact on medicine for diagnosis and treatment of many diseases.
Cloning of DNA
In gene cloning, firstly the DNA of an organism containing the gene of interest in cut into smaller pieces. This gene is called target gene or foreign DNA.

Secondly, the target DNA is joined (in vitro) to a second piece of DNA that can replicate itself and attach any target DNA. This second DNA is often called vector or cloning vehicle. The result of the joining is a hybrid molecule, a hybrid or recombinant DNA
Thirdly, the jointed target and vector is then introduced into a living cell. The cell serves as a biological copying machine, making many exact copies of recombinant molecule. This all process is called molecular cloning.

Tools of gene cloning
Cloning is a basic step in recombinant DNA technology. It involves incorporation of a piece of foreign DNA into a vector. Following tools are needed for cloning of DNA:
a) Restriction endonucleases or enzymes – These enzymes cleave double stranded DNA into smaller fragments. They cut the DNA at specific sites. They are popularly known as molecular scissors.
Restriction enzymes are isolated from bacteria and are named for the bacteria from which they are derived. About hundreds of different restriction enzymes have been isolated. The best known example of a restriction enzyme is EcoR1. These enzymes are highly specific and recognized specific sequences in double-stranded DNA and make two sequence-specific cuts, one in each strand. The most commonly used restriction endonucleases.
b) DNA ligase – DNA ligase is used to covalently link the 3’ – hydroxyl end of one strand of DNA to the 5’ – phosphate ends of second strand. Therefore, DNA segments that are cut by restriction endonucleases can be ligated or joined by DNA ligas enzyme.
c) Vectors – for cloning, a vector is an essential tool in which the foreign gene is inserted. They are also known as cloning vehicles. Vectors must have the following features:
i. They must be able to replicate within the host cell.
ii. They must be capable of insertion into the host cell.
iii. They must have a selectable marker.
iv. They must contain a site for insertion of foreign DNA. In order to carry foreign DNA into cell, the vector must be linked to the foreign DNA.