ANGIOSPERMS

ANGIOSPERMS

1. The term "angiosperms" derives from the two Greek words : angeion meaning "vessels" and sperma meaning "seeds".

2. Angiosperms (flowering plants) are the largest group of plants on earth.

3. Approximately 270,000 known species alive today.

4. Angiosperms include all plants that have flowers and account for approximately 80% of all known living plants.

5. Angiosperms means " covered seeds" have flowers, have fruits with seeds.

Live everywhere- dominants plants in the  world.

6. 260,000 species (88% of plant kingdom).

Angiosperms are most successful and advanced plant on earth.

  

Evolutionary Development of Angiosperms:-

1. Angiosperms evolved during the late Cretaceous period, about 125-100 million years ago.

2. This leaf imprint shows a ficus speciosissiva an Angiosperms that flourished during the Cretaceous period.

3. A large number of pollinating insects also appeared during this same time.

4. Advancements over gymnosperm: angiosperms have flowers- many use pollinators.

5. Fruits and seeds adapted for dispersal.

6. Double fertilization of endosperm in the seed.

Characteristics of Angiosperms

1. Angiosperms have developed flowers and fruits.

2. Flowers serves as the reproductive organs for the plants.

3. Flowers have a wide array of colors, shapes and smells all of the which are for the purpose of attracting pollinators.

1. Roots, stems and leaves.

2. Xylem and phloem.

4. Xylem- conveys water and dissolved minerals from the roots to the shoots.

5. Pholem- transports food made in the leaves to the roots and developing leaves and fruits.

6. They are Heterosporous.

Terminologies:-

a. Androecium- bearing one or more stamens.

b. Gynoecium- bearing one or more carpels.

c. Bisexual (perfect) flower- containing both stamens and carpels.

d. Unisexual (imperfect) flower- having only stamen or carpels.

Distribution of Angiosperms:-

1. One reason for this dominance is the relatively high photosynthetic capacity of their leaves.

2. They occupy every habitat on earth except extreme environment such as the highest mountain tops, the regions immediately surrounding the poles and deepest oceans.

3. They lives as Epiphytes, (i.e. living on other plants).

4. They occur abundantly in-

Shallows of rivers and fresh water lakes.

To a lesser extent, in Salt lakes and in the sea.

 Angiosperms life cycle:-

Flower has male and female sex organs.

Flower structure and it's part:-

1. Male sex organs: stamens, composed of anther- organ that produces pollen (male gametophyte).

2. Female sex organs- the carpel.

3. Ovary is the enlarged basal portion of carpel that contains the ovules (females gametophyte).

4. The stigma is the receptive portionof the carpel for pollen grains to adhere.

5. Non- reproductive parts; sepals (green) are the outermost whorl of leaf like bracts.

6. Petals (usually colored) are the inner whorl of leaf-like bracts.

7. Both can have various shaped and colors.

SEEDS:-

Endosperm is stored food tissues for the embryo to grow. Mature ovule becomes the seed coat and fruits.

Monocot vs Dicot

1. Angiosperms are divided into monocots and dicots.

2. As the zygote grows into the embryo, the first leaves of the young sporophyte develop and are called as cotyledons (seed leaves).

3. Monocots have one cotyledons (corns, lily etc).

4. Dicots have two cotyledons (beans, oak etc).

Comparing Monocots vs Dicots

Economic Importance of Angiosperms

The flowering plants have a number of uses as food, specifically as grains, sugars, vegetables, fruits, oils, nuts and spices.

In addition, plants and their products serve a number of other need such as dyes, fibres, timber, fuel, medicines, and ornaments.

Thank you🙏

BASIC TERMS OF GENETIC

GENETIC

"Genetic is collective study of heredity and variations". 

                          OR

"Genetics is a field of biology that studies how traits are passed from parents to their offspring".

Genetic term is given by William Bateson in the year 1905.

Heredity- Transformation of genetic character from one generation to another.

Variation- Individual of same species have same differences.

There are two types of Variation

1. Somatic variation- they are non inheritable.

2. Germinal/blastogenic variation- they transfer from one generation to another. i.e. inheritable 

a. Continuous (occurs due to crossing over)

b. Discontinuous (occurs due to mutations)

G. J. Mendel is father of genetics. He gave basic principles of genetics in the year 1866.

W. Bateson is father of morden genetics.

Morgan is father of experimental genetics. He did experiments on Drosophila and proposed concept like linkage, sex linkage, crosiing over, criss-cross inheritance, linkage map of Drosophila.

  A. Garrod is father of human genetics and Biochemical genetics. He discovered first human metabolic Disorder, i.e. Alkaptonuria ( Balck urine disease). He gave concept of one-mutant gene one-metabolic block.

TERMS OF GENETICS:-

Gene, allele, locus, homologous chromosomes, Dominant, Recessive, homozygous, heterozygous, gamete formation, phenotype, genotype.

Cell

GENE- A gene is the basic physical and functional unit of heredity that controls a particular trait of an organism.

HOMOLOGOUS CHROMOSOME- At the same locus , same character of gene is present are called homologous chromosomes.

Example- In human  46 chromosomes are present in 23 pairs.

maternal and paternal having same character of gene, at the same location; means both have same gene for eye colour.

LOCUS- Particular location of gene on  chromosomes.

ALLELE-  An allele is an alternate form of same gene, located on same locus on the homologous chromosomes.

                           OR

Allele are the gene which is coding for the same character but not for same trait. (Alternate form of gene)

Character- eye colour.

Trait- Blue, black, brown etc.

Alleles were first defined by Gregor Mendel in the law of segregation.

DOMINANT- A genetic trait is considered dominant if it is expressed in a person who has only one copy of that gene. A dominant trait is opposed to a Recessive trait which is expressed only when two copies of the gene are present.

RECESSIVE- A recessive gene is a gene whose effects are masked in the presence of a Dominant gene. Every organism that has DNA packed into chromosomes has two alleles, or forms of a gene , for each gene ; one inherited from their mother and one inherited from their father. A Recessive gene is only expressed when an organism has two recessive alleles for that gene.

This is also known as Being homozygous recessive. If an organism has one dominant and one recessive allele, it will show the dominant trait.

HOMOZYGOUS- Homozygous is a word that refers to a particular gene that has identical alleles on both homologous chromosome. It is referred to by two capital letters (XX) for a dominant trait, and two small letters (xx) for a Recessive trait.

HETEROZYGOUS- Gene come in pairs, called alleles and each pair is located in a specific position (locus) on a chromosome. If the two alleles at a locus are identical to eachother, they are Homozygous; if they are different from one another, they are Heterozygous.

GENOTYPE- Genetic makeup.

An organism genotype is the set of gene in its DNA responsible for the particular trait.

PHENOTYPE- physical features/apperance.

An organism phenotype is the physical expression of those gene.

                             OR

All the observable characteristics of an organism that results from the interaction of its genotype with the environment. Example- colour, shape, size.

GAMETE FORMATION

Gametes are formed through a process of cell division called meiosis. This two-step division process produces four haploid daughter cells.

Haploid cells contain only one set of chromosomes. When the haploid male and female gametes unite in a process called fertilization, they form zygote. The zygote is diploid and contain two sets of chromosomes.

Thank you👍

                         

GENE THERAPY

GENE THERAPY

Gene therapy is when DNA is introduced into a patient to treat a genetic disease. The new DNA usually contains a functioning gene to correct the effects of a disease-causing mutation.In the future, this technique may allow doctors to treat a disorder by inserting a gene into a patient’s cells instead of using drugs or surgery. Researchers are testing several approaches to gene therapy, including:

• Replacing a mutated gene that causes disease with a healthy copy of the gene.

• Inactivating, or “knocking out,” a mutated gene that is functioning improperly.

• Introducing a new gene into the body to help fight a disease.

Although gene therapy is a promising treatment option for a number of diseases (including inherited disorders, some types of cancer, and certain viral infections), the technique remains risky and is still under study to make sure that it will be safe and effective. Gene therapy is currently being tested only for diseases that have no other cures.

TYPES OF GENE THERAPY

There are two different types of gene therapy depending on which types of cells are treated:

• Somatic gene therapy: transfer of a section of DNA to any cell of the body that doesn’t produce sperm or eggs. Effects of gene therapy will not be passed onto the patient’s children.
• Germline gene therapy: transfer of a section of DNA to cells that produce eggs or sperm. Effects of gene therapy will be passed onto the patient’s children and subsequent generations.

GENE THERAPY WORK

Gene therapy is designed to introduce genetic material into cells to compensate for abnormal genes or to make a beneficial protein. If a mutated gene causes a necessary protein to be faulty or missing, gene therapy may be able to introduce a normal copy of the gene to restore the function of the protein.

A gene that is inserted directly into a cell usually does not function. Instead, a carrier called a vector is genetically engineered to deliver the gene. Certain viruses are often used as vectors because they can deliver the new gene by infecting the cell. The viruses are modified so they can't cause disease when used in people. Some types of virus, such as retroviruses, integrate their genetic material (including the new gene) into a chromosome in the human cell. Other viruses, such as adenoviruses, introduce their DNA into the nucleus of the cell, but the DNA is not integrated into a chromosome.

The vector can be injected or given intravenously (by IV) directly into a specific tissue in the body, where it is taken up by individual cells. Alternately, a sample of the patient's cells can be removed and exposed to the vector in a laboratory setting. The cells containing the vector are then returned to the patient. If the treatment is successful, the new gene delivered by the vector will make a functioning protein.

Researchers must overcome many technical challenges before gene therapy will be a practical approach to treating disease. For example, scientists must find better ways to deliver genes and target them to particular cells. They must also ensure that new genes are precisely controlled by the body.

A new gene is inserted directly into a cell. A carrier called a vector is genetically engineered to deliver the gene. An adenovirus introduces the DNA into the nucleus of the cell, but the DNA is not integrated into a chromosome.

IMPORTANCE OF GENE THERAPY

Genes that are flawed and do not work properly can cause disease. Gene therapy is a technique for correcting defective genes responsible for disease development. Researchers may use one of several approaches for correcting faulty genes:

• A normal gene may be inserted into a nonspecific location within the genome to replace a nonfunctional gene. This approach is most common.
• An abnormal gene could be swapped for a normal gene through homologous recombination.
• The abnormal gene could be repaired through selective reverse mutation, which returns the gene to its normal function.
• The regulation (the degree to which a gene is turned on or off) of a particular gene could be altered.

GENE THERAPY FOR SPECIFIC DISORDERS

Gene therapy holds promise for treating a wide range of diseases, such as cancer, cystic fibrosis, heart disease, diabetes, hemophilia and AIDS. Researchers are still studying how and when to use gene therapy. Currently, in the United States, gene therapy is available only as part of a clinical trial.

Thank you👍

GENE CLONING AND ITS SIGNIFICANCE

DNA cloning 

“DNA cloning is a molecular biology technique which is used for the creation of exact copies or clones of a particular gene or DNA.”

The production of exact copies of a particular gene or DNA sequence using genetic engineering techniques is called gene cloning.

• The term “gene cloning,” “DNA cloning,” “molecular cloning,” and “recombinant DNA technology” all refer to same technique.
• When DNA is extracted from an organism, all its genes are obtained. In gene (DNA) cloning a particular gene is copied forming “clones”.
• Cloning is one method used for isolation and amplification of gene of interest.

DNA cloning can be achieved by two different methods:

1. Cell based DNA cloning
2. Cell-free DNA cloning (PCR)

Requirements of Gene Cloning:-

1. DNA fragment containing the desired genes to be cloned.
2. Restriction enzymes and ligase enzymes.
3. Vectors – to carry, maintain and replicate cloned gene in host cell.
4. Host cell– in which recombinant DNA can replicate.
5. Steps of gene Cloning:
6. The  basic 7 steps involved in gene cloning are:

1. Isolation of DNA [gene of interest] fragments to be cloned.
2. Insertion of isolated DNA into a suitable vector to form recombinant DNA.
3. Introduction of recombinant DNA into a suitable organism known as host.
4. Selection of transformed host cells and identification of the clone containing the gene of interest.
5. Multiplication/Expression of the introduced Gene in the host.
6. Isolation of multiple gene copies/Protein expressed by the gene.
7. Purification of the isolated gene copy/protein.

A. Isolation of the DNA fragment or gene

1. The target DNA or gene to be cloned must be first isolated. A gene of interest is a fragment of gene whose prod­uct (a protein, enzyme or a hormone) interests us. For example, gene encoding for the hormone insulin.
2. The desired gene may be isolated by using restriction endonuclease (RE) enzyme, which cut DNA at specific recognition nucleotide se­quences known as restriction sites towards the inner region (hence endonuclease) producing blunt or sticky ends.
3. Sometimes, reverse transcriptase enzyme may also be used which synthesizes complementary DNA strand of the desired gene using its mRNA.

B. Selection of suitable cloning vector

• The vector is a carrier molecule which can carry the gene of interest (GI) into a host, replicate there along with the GI making its multiple copies.
• The cloning vectors are limited to the size of insert that they can carry. Depending on the size and the application of the insert the suitable vector is selected.
• The different types of vectors available for cloning are plasmids, bacteriophages, bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs) and mammalian artificial chromosomes (MACs).
• However, the most commonly used cloning vectors include plasmids and bacteriophages (phage λ) beside all the other available vectors.

C. Essential Characteristics of Cloning Vectors

All cloning vectors are carrier DNA molecules. These carrier molecules should have few common features in general such as:

• It must be self-replicating inside host cell.
• It must possess a unique restriction site for RE enzymes.
• Introduction of donor DNA fragment must not interfere with replication property of the vector.
• It must possess some marker gene such that it can be used for later identification of recombinant cell (usually an antibiotic resistance gene that is absent in the host cell).
• They should be easily isolated from host cell.

D. Formation of Recombinant DNA

• The plasmid vector is cut open by the same RE enzyme used for isolation of donor DNA fragment.
• The mixture of donor DNA fragment and plasmid vector are mixed together.
• In the presence of DNA ligase, base pairing of donor DNA fragment and plasmid vector occurs.
• The result­ing DNA molecule is a hybrid of two DNA molecules – the GI and the vector. In the ter­minology of genetics this intermixing of dif­ferent DNA strands is called recombination.
• Hence, this new hybrid DNA molecule is also called a recombinant DNA molecule and the technology is referred to as the recom­binant DNA technology.

E. Transformation of recombinant vector into suitable host

• The recombinant vector is transformed into suitable host cell mostly, a bacterial cell.
• This is done either for one or both of the following reasons:
  • To replicate the recombinant DNA mol­ecule in order to get the multiple copies of the GI.
  • To allow the expression of the GI such that it produces its needed protein product.

• Some bacteria are naturally transformable; they take up the recombinant vector automatically.

For example: Bacillus, Haemophillus, Helicobacter pylori, which are naturally competent.

• Some other bacteria, on the other hand require the incorporation by artificial methods such as Ca⁺⁺ ion treatment, electroporation, etc.

F. Isolation of Recombinant Cells

• The transformation process generates a mixed population of transformed and non-trans- formed host cells.
• The selection process involves filtering the transformed host cells only.
• For isolation of recombinant cell from non-recombinant cell, marker gene of plasmid vector is employed.
• For examples, PBR322 plasmid vector contains different marker gene (Ampicillin resistant gene and Tetracycline resistant gene. When pst1 RE is used it knock out Ampicillin resistant gene from the plasmid, so that the recombinant cell become sensitive to Ampicillin.

G. Multiplication of Selected Host Cells

• Once transformed host cells are separated by the screening process; becomes necessary to provide them optimum parameters to grow and multiply.
• In this step the transformed host cells are introduced into fresh culture media .
• At this stage the host cells divide and re-divide along with the replication of the recom­binant DNA carried by them.
• If the aim is obtaining numerous copies of GI, then simply replication of the host cell is allowed. But for obtaining the product of interest, favourable conditions must be provided such that the GI in the vector expresses the product of interest.

H. Isolation and Purification of the Product

• The next step involves isolation of the multiplied GI attached with the vector or of the protein encoded by it.
• This is followed by purification of the isolated gene copy/protein. 
• 

ITS SIGNIFICANCE

• A particular gene can be isolated and its nucleotide sequence determined
• Control sequences of DNA can be identified & analyzed
• Protein/enzyme/RNA function can be investigated
• Mutations can be identified, e.g. gene defects related to specific diseases Organisms can be ‘engineered’ for specific purposes, e.g. insulin production, insect resistance, etc.
• DNA cloning can be used to make proteins such as insulin with biomedical techniques.
• It is used to develop recombinant versions of the non-functional gene to understand the functioning of the normal gene. This is applied in gene therapies also.
• It helps to analyse the effect of mutation on a particular gene.

Thank you👍

Albinism Disorder

Principal of Inheritance and Variations

HUMAN GENETICS DISORDER

An inherited medical condition caused by a DNA abnormality.

Basically two types:-

1. Chromosomal abnormalities

2. Gene Related human disorder

a. Autosomal disorder

      1. Dominant

       2. Recessive

b. Sex (gene disorder)

ALBINISM

 

1. It is autosomal recessive genetic disorder.

2. A group of inherited disorders characterised by little or no melanin production.

3. This condition increases the risk of skin cancer.

4. Albinism Disorder also known as achromasia.

5. Tryosinase enzyme is absent.

6. Individual lack dark pigment melanin in skin, hair and Iris.

Thank you👍

Alkaptonuria Disorder

Principal of Inheritance and variations

ALKAPTONURIA

It is an gene mutation in autosome recessive type. The metabolic defect is the deficiency of homogenticis acid oxidase enzyme. It's an autosomal recessive condition. It is compatible with fairly normal life.

First inborn metabolic disorder described by A. Garrod. 

CAUSE- Defect in the enzyme homogentisate oxidase, that catalysed oxidation of homogentisate to maleylactoactate. 

Homogentisate accumulates in blood and body tissues and is excreted in large amounts in urine.

CHARACTERISTICS FEATURE-

1. The urine of alkaptonuria patients become Dark after being exposed to air.

2. The alkapton imparts a characteristic black-brown color to urine. 

3. Alkaptonuria is a harmless condition.

4. Later in life deposition of dark colored alkapton pigments in connective tissues and bones occur.

5. This results in black pigmentation of the sclera, ear, nose and cheeks and the clinical condition is known as Ochronosis.

6. Ochronosis leads to tissue damage and may develop joint pain, arthritis and backache.

DIAGNOSIS-

1. The urine sample of patients of alkaptonuria turns dark on standing in air.

2. The urine gives positive test with ferric chloride and silver nitrate due to reducing activity of homogentisate.

3. Benedict's test is strongly positive.

TREATMENT-

1. Since alkaptonuria is not considered life threatening, this condition is not treated.

2. Later in life, the Symptoms of arthritis may be treated but the condition itself is not.

3. No specific treatment is required.

4. But minimal protein intake with phenylalanine less than 500gm/day is recommended.

Thank you👍

ETHICAL ISSUES

ETHICAL ISSUES

The manipulation of living organisms by the human race cannot go on any further, without regulation. Some ethical standards are required to evaluate the morality of all human activities that might help or harm living organisms.

Going beyond the morality of such issues, the biological significance of such things is also important.Genetic modification of organisms can have unpredictable result when such organisms are introduced into the ecosystem.

Therefore, the Indian Government has set up organisations 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 granted for the same.

GM crops are already in cultivation in U.S.A. Europe and several other countries. In India, some insect resistant cotton varieties expressing cry genes have reached the farmers, fields. It has been argued that transgenic crops may be harmful to the environment. The two points. Firstly, the transgene may be transferred through pollen from these crops to their wild relatives secondly GM crops may pollute the environment.

BIO-PATENT

A patent is a right granted by a government to an inventor to prevent others from commercial use of his invention A patent is granted for -

1. An invention [including product].

2. An improvement in an earlier invention. 

3. The process of generating products and

4. A concept or design.

There is growing public anger that certain 

 Companies are being granted patents for products and technologies that  make use of the genetic material, plants and other biological resources that have  long been identified, developed and used by farmers and indigenous people of specific region/country.

Rice is an important food grain, the presence of which goes back thousands of years in Asia's agricultural history. There are an estimated 200,000 varieties of tice in lndia alone. The diversity of rice in India is one of the richest in the world, Basmati Rice is distinct for its unique aroma atd flavour and 27 documented varieties of Basmati grown in India. There is reterence to Basmati in ancient texts,folklore and poetry, as it has been grown for centuries. In 1997, an American company got patent rights on Basmati rice through the US Patent and Trademark Office. This allowed abroad This the company to sell a 'new' variety of Basmati in, the US and abroad. The 'New' variety of Basmati had actually been derived from Indian farmers varieties. Indian Basmati was crossed 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 e

medicines e.g. Turmeric neem. If we are not vigilant and we do not immediately counter these patent applications, other countries/individuals may encash on our rich legacy and we may not be able to do anything about it.

BIOPIRACY

Bio-piracy is the term used to refer to the use of bio-resources by multinational companies and other organisations without proper authorisation from the countries and people concemed without compensatory payment.

Most of the industrialised nations are rich financially but poor in biodiversity and traditional knowledge. In contrast the developing and the underdeveloped world is rich in biodiversity and traditional knowledge related to bio-resources. Taditional knowledge related to bio-resources can be exploited to develop modern application and can also be used to save time, effort and expenditure during their commercialisation.

There has been growing realisation of the injustice, inadequate compensation and benefit sharing between developed and developing countries. Therefore, some nations are developing laws to prevent such unauthorised exploitation of their bio-resources and traditional knowledge. 

The Indian Parliament has recently cleared the second amendment the Indian Patents Bill, that takes issues  into consideration, including patent terms emergency provision and research and development initiatives.

Thank you😊

Comment below👇

BIOTECHNOLOGY AND ITS APPLICATION

BIOTECHNOLOGY AND ITS APPLICATION

 

Biotechnology, as you would have learnt  essentially deals with industrial scale production of biopharmaceuticals and biological using genetically modified microbes, fungi, plants and animals. The applications of biotechnology include therapeutics, diagnostics, genetically modified crops for agriculture, processed food, bioremediation, waste treatment and energy production.

(A) BIOTECHNOLOGICAL APPLICATION IN AGRICULTURE :

Let us take a look at the three options that can be thought for increasing food production 

i. agro-chemical based agriculture

ii. organic agriculture and 

iii.  genetically engineered crop-based agriculture. 

The Green Revolution succeeded in tripling the food supply but yet it was not enough to feed the growing human population. Increased yields have partly been due to the use of improved crop varieties, but mainly due to the use of batter management practices and use of agrochemicals (fertilisers and pesticides). However, for farmers in the developing world, agrochemicals are often too expensive and further increases in yield with existing varieties are not possible using conventional breeding is there any alternative path that our undestanding of genetics can show so that farmers may obtain maximum yield from their fields? Is there a way to minimise the use of fertilisers and chemicals so that their harmful effects on the environment are reduced? Use of genetically modified crops is a possible solution.

The Green Revolution succeeded in tripling the food supply but yet it was not enough to feed the growing human population. Increased yields have partly been due to the use of improved crop varieties, but mainly due to the use of better management practices and use of agrochemicals (fertilisers and pesticides). However, for farmers in the developing world, agrochemicals are often too expensive, and further increases in yield with existing varieties are not possible using conventional breeding. Is there any alternative path that our understanding of genetics can show so that farmers may obtain maximum yield from their fields? Is there a way to minimise the use of fertilisers and chemicals so that their harmful effects on the environment are reduced? Uses genetically modified crops is possible solution.

Plants, bacteria, fungi and animals whose genes have been altered by manipulation are called Genetically Modified Organisms (GMO).

GM has been used to create tailor made plants to supply alternative resources to industries, in the form of starches, fuels and pharmaceuticals.

Use of genetically modified (GM) plant:

1. To enhance nutritional quality of food

eg. Golden rice - Vitamin A enriched rice (In this rice gene of B-carotene is transfered).

2. Made crops more tolerant to abiotic stresses (cold, drought, salt, heat).

3. Helped to reduce post harvest losses eg. Flavr Savr Tomato : Transgenic variety of Tomato - Flavr Savr due to the inhibition of polygalacturonase enzyme which degrades pectin. So that tomato variety remains fresh and retain flavour much longer. Flavr Savr Tomato develop by anti-sense technology.

4. Increased efficiency of mineral usage by plants (this prevents early exhaustion of fertility of soil).

5. To produce pharmaceutical product

eg. Production of Hirudin : Hirudin is a protein that prevents blood clotting. The gene incoding hirudin wes chemically synthesized and transfered into Brassica napus. Where hirudin accumulates in seeds. The hirudin is purified and used as medicine.

6. To produce herbicide resistant plant eg. First transgenic plant was tobacco. It contains resistant gene against weedicide (Glyphosate).

7. Pest-resistant crops reduced reliance on chemical pesticides.

i. Insect resistant plant 

eg. Bt cotton : Some strains of Bacillus thuringiensis produce proteins that kill certain insects such as lepidopterans (tobacco budworm, armyworm), coleopterans (beetles) and dipterans (flies, mosquitoes). B.thuringiensis forms protein crystals during a particular phase of their growth. These crystals contain a toxic insecticidal protein.

The Bt toxin protein exist as inactive protoxins but once an insect ingest the inactive toxin, it is converted into an active form of toxin due to the alkaline pH of the gut 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.

Bacillus thuringiensis, produces crystal [Cry] protein. This Cry protein is toxic to Larvae of certain insects. Each Cry protein is toxic to a different group of insects. The gene encoding cry protein is called "cry gene". This Cry protein isolated and transferred into several crops. A crop expressing a cry gene is usually resistant to the group of insects for which the concerned Cry protein is toxic. There are a number of them, for example, the proteins encoded by the genes crylAc and cryllAb control the cotton bollworms, that of crylAb controls corn borer.

However, gene symbol italics, e.g., cry. The first letter or the protein symbol, on the other hand is always capital and the symbol is always written in roman letters. e.g., Cry.

ii. Nematode resistant plant:

Several nematodes parasitise a wide variety of plants and animals including human beings. A nematode Meloidogyne incognitia infects the roots of tobacco plants and causes a great reduction in yield. A novel strategy was adopted to prevent this infestation which was based on the process of RNA Interference (RNAi). RNAi takes place in all eukaryotic organisms as a method of cellular defense.

This method involves silencing of a specific mRNA due to a complementary dsRNA molecule that binds to and prevents translation of the mRNA (silencing). The source of this complementary RNA could be from an infection by viruses having RNA genomes or mobile genetic elements (transposons) that replicate via an RNA intermediate.

Using Agrobacterium vectors, nematode-specific genes were introduced into the host plant. The introduction of DNA was such that it produced both sense and antisense RNA in the host cells. These two RNA's being complementary to each other formed a double stranded (dsRNA) that initiated RNAi and thus, silenced the specific mRNA of the nematode. The consequence was that the parasite could not survive in a transgenic host expressing specific interfering RNA. The transgenic plant therefore got itself protected from the parasite.

BIOTECHNOLOGICAL APPLICATIONS IN MEDICINE

A. Genetically Engineered Insulin

It is a proteinaceous hormone having 51 Amino acids arranged in two polypeptides A and B having 21 and 30 Amino Acids respectively and joined by S-S disulphide

bridges.

Sir Edward sharpy-Shafer (1916) was the first to note that diabetes of some persons was because of failure of some islands of pancreas to produce a substance which he called insulin (Derived from the latin, insula, meanning island).

Banting and best (1921) were the first to isolate insulin from dog's pancreas and used it to cure diabetes in man. The first genetically engineered insulin obtained by recombinant DNA technique with the help of E-Coli was produced by the American firms, Eli-Lilly on July 5, 1983. It has been given the trade name humulin and has been approved for clinical use.

Insulin used for diabetes was earlier extracted from pancreas of slaughtered cattle and pigs. Insulin from an animal source, though caused some patients to develop allergy or other types of reactions to the foreign protein. Insulin consists of two short polypeptide chains: chain A and chain B, that are linked together by disulphide bridges. In mammals, including humans, insulin is synthesised as a prohormone (like a pro-enzyme,the pro-hormone also needs to be processed before it becomes a fully mature and functional hormone) which contains an extra stretch called the C peptide.

 This C peptide is not present in the mature insulin and is removed during maturation into insulin. The main challenge for production of insulin using rDNA techniques was getting insulin assembled into a mature form. In 1983, Eli Lilly an American company prepared two DNA sequences corresponding to A and B, chains of human insulin and introduced them in plasmids of E.coli to produce insulin chains. Chains A and B were produced separately, extracted and combined by creating disulfide bonds to form human insulin.

maturation of pro-insulin after removal of C-peptide (to be simplified)

B.Gene Therapy: 

A new system of medicine gene therapy, may develop to treat some hereditary diseases such as SCID, haemophilia etc.

Gene therapy is a collection of methods that allows correction of a gene defect that has been diagnosed in a child/embryo. Here genes are inserted into a person's cells and tissues to treat a disease. Correction of a genetic defect involved delivery of a normal gene into the individual or embryo to take over the function of and compensate for the non-functional gene.

The first clinical gene therapy was given in 1990 to a 4-year old girl with adenosine deaminase (ADA) deficiency. This enzyme is crucial for the immune system to function. The disorder is caused due to the deletion of the gene for adenosine deaminase. In some children ADA deficiency can be cured by bone marrow transplantation, in others it can be treated by enzyme replacement therapy, in which functional ADA is given to the patient by injection. But the problem with both of these approaches that they are not completely curative. As a first step towards gene therapy, lymphocytes from the blood of the patient are grown in a culture Outside the body. A functional ADA cDNA (using a retroviral vector) is then introduced into these lymphocytes.

which are subsequently returned to the patient. However, as these cells are not immortal, the patient requires periodic infusion of such genetically engineered lymphocytes. However, if the gene isolate from marrow cells producing ADA is introduced into cells at early embryonic stages, it could be a permanent cure.

 C. Medical Diagnosis of Disease (Molecular diagnosis) 

You know that for effective treatment of a disease, early diagnosis and understanding its pathophysiology is very important. Using conventional methods of diagnosis (serum and urine analysis, etc.) early detection is not possible. Recombinant DNA technology, Polymerase Chain Reaction (PCR) and Enzyme Linked Immuno- sorbent Assay (ELISA) are some of the techniques that serve the purpose of early diagnosis.

Presence of a pathogen (bacteria, viruses, etc) is normally suspected only when the pathogen has produced a disease symptom. By this time the concentration of pathogen is already very high in the body. However, very low concentration of a bacteria or virus (at a time when the symptoms of the disease are not yet visible) can be detected by amplification of their nucleic acid by PCR. PCR is now routinely used to detect HIV in suspected AIDS patients. It is being used to detect mutations in genes in suspected cancer patients too. It is a powerful technique to identify any other genetic disorders.

A single stranded DNA or RNA, tagged with a radioactive molecule (probe) is allowed to hybridise to its complementary DNA in a clone of cells followed by detection using autoradiography. The clone having the mutated gene will hence not appear on the photographic film, because the probe will not have complimentarity with the mutated gene.

ELISA is based on the principle of antigen-antibody interaction. Infection by pathogen can be detected by the presence of antigens (proteins, glycoproteins, etc.) or by detecting the antibodies synthesised against the pathogen.

TRANSGENIC ANIMALS

Animals that have had their DNA manipulated to possess and express an extra (foreign) gene are known as transgenic animals. Transgenic rats, rabbits, pigs, sheep, cows and fish have been produced, although over 95 percent of all existing transgenic animals are mice.

1. Normal physiology and development: Transgenic animals can be specifically designed to allow the study of how genes are regulated, and how they affect the normal functions of the body and its development e.g. study of complex factors involved in growth such as insulin-like growth factor. By intoducing genes from other species that alter the formation of this factor and studying the biological effects that result, information is obtained about the biological role of the factor in the body.

2. Study of disease: Many transgenic animals are designed to increase our understanding of how genes contribute to the development of disease. These are specially made to serve as models for humane diseases so that investigation of new treatments for diseases is made possible. Today transgenic models exist for many human diseases, such as cancer, cystic fibrosis, rheumatoid arthritis and Alzheimer's.

3. Biological products : Medicines required to treat certain human diseases can contain biological products, but such products are often expensive to make. Transgenic animals that produce useful biological products can be created by the introduction of the portion of DNA (or genes) which codes for a particular product such as human protein ( α-1-antitrypsin) used to treat emphysema, Similar 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 milk contained the human alpha-lactalbumin and was nutritionally a more balanced product for human babies that natural cow-milk.

4. Vaccine safety : Transgenic mice are being developed for use in testing the safety of vaccines 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: This is known as toxicity/safety testing. The procedure is the same as that used for testing toxicity of drugs. Transgenic animals are made that carry genes which make them more sensitive to toxic substances than non-transgenic animals. They are then exposed to the toxic substances and the effects studied. Toxicity testing in such animals will allow us to obtain results in less time.

Note:-

1. First transgenic animal was mouse contain gene for growth hormone. This enlarged mouse was known as supermouse.

2. First introduced transgenic crop in India (2002) is Bt-cotton.

3. Charles weismann of University of Zurich, obteined interferon through recombinant E. Coli (1980).

4. Microbe have been engineered to produce Human growth Hormone (HGH) for curing dwarfism.

5. Vaccines which are produced by genetic engineering e.g., for Hepatitis-B and Herpes virus.

6. Nitrogen fixation genes may be transferred from bacteria to the major food crops to boost food production without using expensive fertilizers.

7. Bioremediation: in pollution control, microbes have been engineered to break up the crude oil spills. Dr. Ananda Mohan chakarborthi introduced plasmids from different strains into a single cell of Pseudomonas putida. The  result was new genetically engineered bacterium which would clean the oil spills called "Superbug" (Oil eating bug). He transferred four types of genes/ plasmids in this bacteria. These are OCT, XYL, CAM and NAH.

8. Genetic modified food- the food is prepared from genetically modified crop (transgenic) is called genetically modified food or G.M. Food.

Thank you👍

BIOTECHNOLOGY- PRINCIPLES AND PROCESSES

BIOTECHNOLOGY - PRINCIPLES & PROCESSES

Biotechnology term given by Karl Ereky. Biotechnology deals with techniques of using live organisms or enzymes from organisms to produce products and process useful to humans.

 Types of Biotechnology Two types- 

(1) Old/traditional- 

Old biotechnology are based on the natural capabilities of micro organisms.

e.g. formation of Citric acid, production of penicillin by Penicillium notatum, making curd bread or wine, which are all microbe mediated processes a form of biotechnology.

(2) New/modern-

Based on Recombinant DNA technology.

e.g Human gene producing Insulin has been transferred and expressed in bacteria like E.coli.

 According to the European Federation of Biotechnology (EFB) has given a definition of biotechnology that encompasses both traditional view and modem molecular biotechnology. The definition given by EFB is as  follows: 'The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services'.

Paul berg (Father of genetic engineering)-  He transferred gene of SV-40 virus (simian virus) in to E.coli with the help of  -phage (Nobel prize 1980).

Stanley Cohen and Herbert Bover: First made recombinant DNA by linking an  antibiotic resistance gene with a native plasmid of Salmonella typhimurium.

Principles of Biotechnology 

Among many, the two core techniques that enabled birth of modern biotechnology are:-

1. Genetic Engineering/Recombinant DNA Technology:

 Techniques to alter the chemistry of genetic material (DNA and RNA), to introduce these into host organisms and thus change the phenotype of the host organism. (i.e. formation  of genetically modified organism).

2. Bioprocess Technology:

Maintenance of sterile (microbial contamination free) ambience in chemical engineering processes to enable growth of only the desired microbe/eukaryotic cell in large quantities for the manufacture biotechnological products like antibiotics, vaccines.enzymen, etc.

The concept of genetic engineering was the outcome of two very significant discoveries made in bacterial research. These were-

1. presence of extrachromosomal DNA fragments called plasmids in the bacterial cell, which replicate along with chromosomal DNA of the bacterium.

2. Presence of enzymes restriction endonucleases which cut DNA at specific sites. These enzymes are, Therefore, called 'molecular scissors'.

The main basis of Recombinant DNA Technology is DNA cloning:- it is  making multiple identical copies of any template DNA.

There are three basic steps of DNA clonning-

i) identification of DNA with desirable genes.

ii) introduction of the identified DNA into the host.

iii) maintenance of introduced DNA in the host and transfer of the DNA to its progeny.

TOOLS OF RECOMBINANT DNA TECHNOLOGY :

Genetic engineering involves cutting of desired segments of DNA and pasting of this D.N.A in a vector to produce a recombinant DNA (rDNA). The 'biological tools' used in the synthesis of recombinant DNA include enzymes, vehicle or vector DNA, desired DNA and host cells.

1. Enzymes :- A number of specific kinds of enzymes are employed in genetic engineering.

These include lysing enzymes, cleaving enzymes, synthesising enzymes and joining enzymes.

Lysing enzymes - These enzymes are used for opening the cells to get DNA for genetic experiment.

Bacterial cell : is commonly digested with the help of lysozyme.

Plant cell : is commonly digested with the help of cellulase and pectinase

• Fungal cell : is commonly digested with the help of chitinase.

Cleaving enzymes - These enzymes are used for DNA molecules. Cleaving enzymes are of two types: exonucleases and endonucleases.

Exonuclease remove nucleotides from the ends of the DNA.

Endonucleases make cuts at specific positions within the DNA.

eg. Restriction endonucleases

Restriction Endonuclease Enzymes (Molecular scissors or molecular knife) Bestriction enzymes belong to a larger class of enzymes called endonucleases.

Restriction enzymes are used in recombinant DNA Technology because they can be used in vitro to recognize and cleave within specefic DNA sequence typically consisting of 4 to 8 nucleotides. This specie 4 to 8 nucleotide sequence  is called restriction site and is usually palindromic, this means that the DNA sequence is the same when read in a 5'-3' direction on both DNA strand.

Restriction enzymes are obtained from bacteria.

 What is function of restriction enzymes in bacteria?

They are useful to bacteria because the enzyme bring about fragmentation of viral DNA without affecting the bacterial genome. This is an adaptation against bacteriophages. These enzymes exist in many bacteria beside cleavage some restriction on endonuclease, also have capability of modification.

Modification in the form of methylation, by methylation the bacterial DNA modifies and therefore protects its an own chromosomal DNA from cleavage by these restriction enzymes.

 The first restriction endonuclease-Hind II, whose functioning depended on a specific DNA nucleotide sequence was  isolated and characterised five years later. It was found that Hind II always cut DNA molecules at a particular point by recognising a specific sequence of six base pairs. This specific base sequence is known as the recognition sequence for Hind II.

Restriction enzyme (Eco R-I) was discovered by Arter Smith & Nathans (1978 Nobel prize).

Besides Hind II,  today we know more thán 900 restriction enzyme that have been isolated from over 230 strains of bacteria each of which recognise different recognition sequences.

Nomenclature of enzyme -

The first letter- indicates bacterial genus (In italic).

 Second and third letter- indicate species of bacteria (in italics).

 Fourth letter- indicates strain of bacteria (optional).

 Roman numeraical- signifying the order in which the enzymes were Isolated from that strain of bacteria.

eg. EcoRI comes from Escherichia coli RY 13. In EcoRL the letter 'R' is derived from the name of strain. Roman numbers following the names indicate the order in which the enzymes were isolated from that strain of bacteria.

Restriction enzymes forms two types of ends on the basis of mode of cutting.

Sticky end (Free end) : Restriction enzymes cut the strand of DNA a little away from the centre of the palindrome sites, but between the same two bases on the opposite strands.

This leaves single  stranded portion at the ends. There are overhanging stretches called sticky ends on each. These are named so because they form hydrogen bonds with their complementary cut counterparts. This stickiness of the ends facilitates the action of the enzyme DNA ligase.

eg. EcoRI, Hind III, Bam HI, Sal l.

Blunt end (non sticky end) : some enzymes cleave both strand of DNA at exactly the same nucleotide position, typically in the center of the recognition sequence resulting in blunt end or flush end.

eg. Sma l, EcoRV, Hae III.

 Sma l (Serratia marcescens)

Synthesizing enzymes- These enzymes are used to synthesize new strands of DNA, complementary to existing DNA or RNA template. They are of two types; reverse transcriptases and DNA polymerases.

Reverse transcriptases help in the synthesis of complementary DNA strands on RNA templates.

DNA polymerases- help in the synthesis of complementary DNA strands on DNA templates. 

Joining enzymes- These enzymes help in joining the DNA fragments. For example DNA ligase from Escherichia coli is used to join DNA fragments. Joining enzymes are, therefore, called molecular glues.

2. Vehicle DNA or Vector DNA-  The DNA used as a carrier for transferring a fragment of foreign DNA into a suitable host is called vehicle or vector DNA.

You know that plasmids and bacteriophages have the ability to replicate within bacterial cells independent of the control of chromosomal DNA.

The following are the features that are required to facilitate cloning into a vector.

A. Origin of replication (ori) : This is a sequence from where replication starts and any piece of DNA when linked to this sequence can be made to replicate within the host cells. This sequence is also responsible for controlling the copy number of the linked DNA. So, if one wants to recover many copies of the target DNA it should be cloned in a vector whose origin support high copy number.

B. Selectable marker: In addition to 'ori', the vector requires a selectable marker, which helps in identifying and eliminating non transformants and selectively permitting the growth of the transformants.

• Normally, the genes encoding resistance to antibiotics such as ampicillin, chloramphenicol, tetracycline or kanamycin, etc. are considered useful selectable markers for E.coli

• Enzyme forming gene also act as selectable marker gene, eg Lac. Z gene Note: The normal E. coli cells do not carry resistance against any of these antibiotics.

C. Restriction sites/Cloning sites : In order to link the alien DNA, the vector needs, recognition sites for the commonly used restriction enzymes. The ligation of alien DNA is carried out at a restriction site present in one of the two antibiotic resistance genes.

A vector should have restriction site for many enzyme but only one restriction site for each enzyme, Otherwise vector will get fragmented.

Selection of vector depends on :-

1. Size of desired gene 

2. Type of host 

Some examples of vectors:-

a. Plasmids- They are double stranded, circular and extra chromosomal DNA segments found in bacteria which can replicate independently. Plasmids can be taken out of bacteria and made to combine with desired DNA segments by means of restriction enzymes and DNA ligase. A plasmid carrying DNA of another organism integrated with it, is known as recombinant plasmid or hybrid plasmid or Chimeric plasmid.

eg. pBR 322, PUC 18 (used for gene transfer in bacteria).

Ti plasmid, Ri Plasmid (used for gene transfer in dicot plant).

b. Viruses- The DNA of certain viruses is also suitable for use as a vehicle DNA.

Bacteriophage DNA also used as a vector DNA for gene transfer.

Lambda phage (λ phage) has been used for transferring lac genes of E. coli into haploid callus of tomato.

Retro virus - useful for gene transfer in animal cell.

3. Desired DNA/ Alien DNA/ Foreign DNA/ Passenger DNA:-

It is the DNA which is transferred from one organism into another by combining it with the vehicle DNA. The passenger DNA can be complementary, synthetic or random.

i. Complementary DNA (cDNA)- It is synthesized on mRNA template with the help of reverse transcriptase and necessary nucleotides. cDNA formed through reverse transcription is shorter than the actual or in vivo gene because of the absence of introns or non-coding regions.

ii. Synthetic DNA (sDNA)- It is synthesized with the help of DNA polymerase on DNA template.

Kornberg (1961) synthesized first synthetic DNA.

Khorana (1968) synthesized first artificial gene (DNA) without a template. They synthesized the gene coding for yeast alanine t-RNA, which contained only 77 base pairs. However, it did not function in the living system. In 1979, Khorana was able to synthesis a functional tyrosine t-RNA gene of E. coli with 207 nucleotide pairs. Since then a number of genes have been synthesized artificially.

4. Host organism :- 

This organism is used for DNA cloning. Host may be plant, animal, bacteria (E.col. fungi (Yeast).

PROCESSES OF RECOMBINANT DNA TECHNOLOGY:

1. Isolation of DNA: The DNAs which are to be used as passenger DNA and the vehicle DNA are extracted out of their cells by lysing  the cells with the suitable enzyme.

The DNA is enclosed within the membranes, we have to break the cell open to release DNA along with other macromolecules such as RNA, proteins, polysaccharides and also lipids. This can be achieved by treating the bacterial cells/plant or animal tissue with enzymes such as lysozyme (bacterial), cellulose (plant cells), chitinase (fungus). You know that genes are located on long molecules of DNA intertwined with proteins such as histones.

The RNA can be removed by treatment with ribonuclease whereas proteins can be removed by treatment with protease. Other molecules can be removed ty appropriate treatments and purified DNA ultimately precipitates out after the addition of chilled ethanol. This can be seen as collection of fine threads in the suspension DNA that separates out can be removed by spooling method.

2. Fragmentation of DNA by restriction endonucleases : Restriction enzyme digestions are performed by incubating purified DNA molecules with the restriction enzyme, at the optimal conditions for that specific enzyme. Agarose gel electrophoresis is employed to check the progression of a restriction enzyme digestion.

Both the passenger and vehicle DNAs are then, cleaved by using the same restriction endonuclease so that they have complementary sticky ends.

Note : The separated DNA fragments can be visualised only after staining the DNA with a compound known as ethidium bromide followed by exposure to UV radiation (you cannot see pure DNA fragments in the visible light and without staining). You can see bright orange coloured bands of DNA in a ethidium bromide stained gel exposed to UV light. The separated bands of DNA are cut out from the agarose gel and extracted from the gel piece. This step is known as elution. The DNA fragments purified in this way are used in constructing recombinant DNA by joining them with cloning vectors.

3. Amplification of gene of interest using PCR

Polymerase chain reaction technology (PCR-technology)

This technique was invented by Kary mullis (1983).

In 1993 Kary Mullis got nobel prize for PCR (for chemistry).

PCR is a method for amplifying a specific region of DNA molecule without the requirement for time consuming cloning procedures.

PCR reaction takes place in Eppendorf tube.

Using PCR-technique very low content of DNA available from samples of blood or semen or any other tissue or hair cell can be amplified many times and analysed. In this technique Taq-Polymerase is used. Taq polymerase enzyme is used in PCR which is a special type of DNA polymerase enzyme which is resistant to high temperature.

Taq Polymerase is isolated from Thermus aquaticus bacteria.

Some other examples of polymerase which are used in PCR are-

Pflu Polymerase - Isolated from Pyrococcus furiosus bacteria.

Vent Polymerase - Isolated from Thermococcus litoralis bacterium

Main steps in PCR :-

Denaturation (94°) :- In this step a double stranded DNA molecule is placed at 94°C. So double stranded DNA becomes single stranded & each single stranded DNA functions as a template.

Annealing/Cooling (54°) - In this step two primer DNA are attached at 3' end of single stranded DNA.

Extension (72°):- In this process Taq polymerase enzyme synthesize DNA strain over template.

PCR is automatic process because Taq. polymerase enzyme is heat resistant.

4. Ligation of the DNA fragment into a vector- The complementary sticky ends of the passenger and vehicle DNAs are joined with ligase enzyme. This gives rise to a recombinant DNA.

5. Transferring the recombinant DNA into the host (Gene transfer):-

Transfer of desired genes from one organism into another is an important aspect of genetic engineering.

Gene transfer is achieved by two kinds of transfer methods:

1. Indirect method through vectors or carriers and

2.  Director vector less transfer method.

Indirect method :-

Gene Transfer in bacterial cell : Since DNA is a hydrophilic molecule, it cannot pass through cell membranes. In order to force bacteria to take up the plasmid, the bacterial cells must first be made 'competent' to take up DNA. This is done by treating them with a specific concentration of a divalent cation, such as calcium, which increases the efficiency with which DNA enters the bacterium through pores in its cell wall. Recombinant DNA can then be forced into such cells by Incubating the cells with recombinant DNA on ice, followed by placing them briefly at 42°C (heat shock), and then putting them back on ice. This enables the bacteria to take up the recombinant DNA.

Note: The bacteria to be used as hosts should be without plasmids. The host cells are treated with calcium chloride or lysozyme.

Transformant E.coli with plasmid.

Non-transformant E.coli without plasmid.

Gene Transfer in plant cell 

1. A plant pathogenic bacterium-Agrobacterium tumefaciens produces crown galls or plant tumours in almost all dicotyledonous plants.

2. This bacterium infects all broad leaved agricultural crops such as tomato, soyabean, sunflower and cotton but not cereals.

3. Tumour formation is induced by it's plasmid which is therefore called Ti plasmid (TI - tumour inducing) Agrobacterium tumefaciens naturally transfers some part of Ti-Plasmid into host plant DNA without any human effort so it is called natural genetic engineer of plant.

In the transformation process two essential component in Ti-plasmid -

 (a) T-DNA - (Transferred DNA)

(b) Vir-region-(Virulence region)

Inside the host plant cell T-DNA is seperated from Ti-plasmid, and integrated into host plant DNA that causes crown gall tumour.

Vir-region contains genes which are essential for T-DNA transfer and integration to host plant DNA.

When we use Ti-plasmid as a vector, first we remove the tumour causing gene from T-DNA region. Then desired gene inserted inplace of it. Now, this plasmid is called disarmed plasmid.

Same as Ri plasmid of A.rhizogenes (causing hairy root disease) also used as vector for gene transfer to plant cell.

Gene transfer in animal

Retroviruses have also been disarmed and are now used to deliver desirable genes into animal cells.

Direct method

Foreign genes can also be transferred directly by the following methods:-

a. Electroporation- It creates transient (temporary pores) in the plasma membrane to facilitate entry of foreign DNA.

b. Chemical mediated genetic transformation- It involves certain chemicals such as polyethylene gycol (PEG), that help in the uptake of foreign DNA into host cells.

c. Microinjection- it in the introduction of foreign genes mainly into animal cells using micropipette ar glass needles.

d. Particle gun/Biolistic method- It is a technique in which tungsten or gold particles coated with foreign DNA bombarded into target cells to facilitate entry of the foreign genes.

e. Liposome mediated gene transfer- In this method DNA encloses within lipid begs. Thest lipid begs funed with protoplast.

Selection of Transformed with recombinant cell:-

1. Selection by two antibiotic resistance gene 

You can ligate a foreign DNA at the Bam HI site of tetracycline resistance gene in the vector pBR322.

The recombinant plasmids will loss tetracycline resistance due to insertion of foreign DNA (insertional inactivation) now, it can be selected out from non-recombinant ones by plating the transformants on ampicillin containing medium. The transformants (plasmid transferd) growing on ampicillin containing medium are then transferred on a medium containing tetracycline. The recombinants will grow in ampicillin containing medium but not on that containing tetracycline. But, non recombinants wil grow on the medium containing both the antibiotics. In This case, one antibiotic resistance gene helps in selecting the transformants.

Note:- 

Insertional inactivation- Due to Insertion of desired gene within selectable marker gene of vector, selectable marker gene become inactive or loose their Function. This is called Insertional inactivation.

2. Selection by one Lac Z gene and one antibiotics resistant gene-   Selection of recombinants due to inactivation of antibiotics is a cumbersome (troublesome) procedure because it requires simultaneous plating on two plated having different antibiotics. Therefore, alternative selectable markers have been developed which differentiate recombinants from non-recombinants on the basis of their ability to produce colour in the presence of a chromogenic substrate. In this, a recombinant DNA is inserted within the coding sequence of an enzyme, which is referred to as insertional inactivation. The presence of a chromogenic substrate X-gal gives blue coloured colonies if the plasmid in the bacteria does not have an insert. Presence of insert results into insertional inactivation of the β-galactosidase (reporter enzyme) and the colonies do not produce any colour, these are identified as recombinant colonies.

Obtaining the Foreign Gene Product :

When you insert a piece of alien DNA into a cloning vector and transfer it into a bacterial plant or animal cell the alien DNA gets multiplied. In almost all recombinant technologies, the ultimate aim is to produce a desirable protein. Hence, there is a need for the recombinant DNA to be expressed. The foreign gene gets expressed under appropriate conditions. The expression of foreign genes in host cells involve understanding many technical details.

After having cloned the gene of interest and having optimised the conditions to induce the expression of the target protein, one has to consider producing it on a large scale. Can you think of any reason why there is a need for large-scale production? If any protein encoding gene is expressed in a heterologous host, is called a recombinant protein. The cells harbouring cloned genes of interest may be grown on a small scale in the laboratory. The cultures may be used for extracting the desired protein and then purifying it by using different separation techniques.

The cells can also be multiplied in a continuous culture system wherein the used medium is drained out from one side while fresh medium is added from the other to maintain the cells in their physiologically most active log/exponential phase. This type of culturing method produces a larger biomass leading to higher yields of desired protein.

Small volume cultures cannot yield appreciable quantities of products. To produce in large quantities, the development of bioreactors, where large volumes (100-1000 litres) of culture can be processed, was required. Thus, bioreactors can be thought of as vessels in which raw materials are biologically converted into specific products, individual enzymes, etc. useing microbial plant, animal or human cells. A bioreactor provides the optimal conditions for achieving the desired product by providing optimum growth conditions (temperature, pH, substrate, salts, vitamins, Oxygen).

The most commonly used bioreactors are of stirring type -

A stirred-tank reactor is usually cylindrical or with a curved base to facilitate the mixing of the reactor contents.

The stirrer facilitates even mixing and oxygen availability throughout the bioreactor. Alternatively, air can be bubbled through the reactor. The bioreactor has an agitator system, an oxygen delivery system and a 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.

Thank you👍