BIOTECHNOLOGY AND GENETIC ENGINEERING

The use of scientific and engineering principles to manipulate organisms or their genes is Biotechnology. The classical biotechnology involves selection hybridization and mutation. For more than 8000 years bacteria yeast have been used to produce beers, vinegar, yogurt and cheese. Since ancient times, plants and cheese. Since ancient times, plants and animals breeders have selectively bred organisms to develop hybrids. In the late 1920s, scientists induced genetic mutation and obtained plants with new characteristics. The United Nations Convention on Biological Diversity (UNCBD) defines biotechnology as:

“Any technological application that uses biological systems living organisms or derivatives thereof, to make or modify products or processes for specific use”. In other words “Application of scientific and technical advances in life sciences to develop commercial products” is biotechnology. A revolution took place in this area after the discovery of molecular structure of the hereditary material deoxyribo-nucleic acid (DNA).Another important landmark is the discovery of restriction enzymes. These are made by bacteria as a defense mechanism against viruses. Restriction enzymes bind to specific sites (restriction sites) of viral DNA and restrict the growth of virus by cutting the DNA into small fragments (fig.B.3.5). these enzymes can be used to cut any DNA to produce small fragments of DNA, which can be separated by size using Gel electrophoresis. Specific DNA sequence can be identified in a gel by probes with a complementary sequence using Southern Blotting technique.

Blotting technique.

  The action of restriction endonuclease (EcoR I) of DNA. The enzymes make sticky ends which helps to make recombinant DNA.

Recombinant DNA is formed by the combination of two DNA from different source. One of the major goals of recombinant DNA technology is to clone a particular gene. Bacteria, yeasts and cultured plant cells are commonly used as hosts for recombinant DNA. Host cells into which recombinant DNA is inserted, or transformed, are called transgenic cells. To identify host cells that have taken up a foreign gene, the inserted sequence can be tagged with reporter genes genetic markers with easily identifiable phenotypes. Expression of the foreign   gene in the host cell requires, so that it can become the part of a segment of DNA, a replicon (origin and terminus of replication). There are two general ways in which the newly introduced DNA can become part of a replicon.

It can be inserted near on origin of replication in a host chromosome after entering the host cell.

It can enter the host cell as a part of a carrier DNA sequence called vector that already has the origin of replication.

 Plasmids, viruses and artificial chromosome (YAC – yeast artificial chromosome, BAC- Baxterial artificial chromosome) are all used as vector. DNA for cloning can be obtained from gene libraries, cDNA made from mRNA, and artificial synthesized DNA fragments.

   Biotechnology has applications in four major industrial areas, including health care, crop producer products (e.g., biodegradable plastics, vegetables oil, biofuel) and environmental uses.

The several branches of biotechnology are:

Bioinformatics: It is also referred to as computational biology I and defined as “conceptualizing biology, in terms of molecules and then applying informatics techniques to understand and organize the information associated with these molecules, on a large scale”. It is an interdisciplinary field which solves biological problems using computational techniques and analysis of biological data. It plays a key role in functional genomics, structural genomics and proteomics and forms a key component in the biotechnology and pharmaceutical sector.

Blue Biotechnology: The term uage is relatively rare. It is used to describe the marine and aquatic applications of biotechnology.

Green biotechnology: It is the application of biotechnology to agricultural processes. It includes micro- propagation, transgenic plants and industrial agricultural. An example of this is the engineering of a plant to express a pesticide (Bt cotton).

Red biotechnology: It is applied to medical processes. Designing of organisms to produce antibiotics, and the engineering of genetic cures through genetic manipulation.

White Biotechnology: It also known as industrial biotechnology. It involves the use of enzymes as industrial catalysts to either produce valuable chemicals or destroy hazardous polluting chemicals

  The investment and economic output tall these types applied bio technologies is termed the bio-economy.

Genetic engineering: Human has alerted the genomes of species for thousands of years through artificial selection. In 1986, tobacco plants were engineered to be resistant to herbicides and the first field trials of genetically engineered plants we done. The basic steps involved in genetic engineering are (Fig.B.3.6)

·         Isolation of the gene: The gene to be interested into the genetically modified organism must be chosen and isolated. Most of the genes transferred into plants provide protection against insect or tolerance to herbicide. The gene can be isolated from gene library or artificially synthesized. The gene is then inserted into bacterial plasmid.

·         Cloning Vectors: The gene is inserted into other genetic elements for its proper functioning. The gene to be inserted must contain a promoted and terminator region as well as a selectable marker gene. The promoter region initiates transcription of the gene, while the terminator region ends transcription. The selectable marker, antibiotic resistance, helps to determine which cells are transformed with the new gene. The constructs are made using recombinant DNA techniques, such as restriction digest, ligations and molecular cloning.

·         Gene Targeting: The insertion of new genetic material randomly within the horst genome. The frequency of gene targeting can be enhanced by the use of engineered nucleases such as zinc finger.

        Genetic Transformation: DNA is generally inserted into animal cells using microinjection or through the use of viral vectors. In plants the DNA is generally inserted using Agrobacterum –mediated recombination or biolistics. Agrobacterium naturally inserts DNA T-DNA from a tumor inducing (Ti) plasmid into any susceptible plant’s geneome it infects, causing crown gall disease. The T-DNA region of this plasmid is responsible for insertion of the DNA. The genes to be inserted are cloned into a binary vector, which contains T-DNA and can be grown in both E-coli and Agrobacterium. The Agrobacterium naturally insert the genetic material into the plant cells. In biolistics, particles of gold or tungsten are coated with DNA and then shot into young plant cells or embryos. Another method for plant and animal cells is electroporation; involves subjecting the plant or animal cell to an electric shock, which can make the cell membrane permeable to plasmid

Selection: By growing the cells in the presence of an antibiotic or chemical that selects or marks the cell expressing that gene it is possible to separate the transgenic events from the non-transgenic. DNA probe is also used for screening; it only sticks to inserted gene  

 Regeneration: In plants, tissue culture methods are used to regenerate from single transformed cells. In animals, the inserted DNA is present in the embryonic stem cells.

All the offspring from the first generation will be heterozygous for the inserted gene and must be mated together to produce a homozygous animal.

There are other newly developed tools which are used to manipulate DNA.

·         Gene Knockout: It is a technique in which one of the genes in the organism is made inoperative. This technique is used for learning gene function. The difference in normal and knockout individuals tell the role of knockout gene. The first knockout mouse was created by Mario R Capecchi, Martin Evans and Oliver Smithies in 1989, for which they were awarded the Nobel prize for medicine in 2007. A knockout mouse is genetically engineered one in which inactivated or knockout gene is replaced by artificial piece of DNA. The loss of gene activity causes changes in the mouse. These are important animal models for studying the role of a gene.

·         Gene silencing: Gene silencing involves switching off of the genes by a mechanism switching off of the gene modification. It can be used to inactivate the mRNA transcript of a gene. The mRNA can be blocked by a complementary RNA. The blocking RNA is known as artificial antisense RNA and interference RNA. Once blocked the single stranded mRNA becomes double stranded. The double stranded RNA does not allow the protein synthesis to take place. The gene has been silenced by blocking the mRNA at transcriptional level. This technique is useful in treating genetic disorders. A defective gene can be stopped to function in an organism.

·         DNA chips: DNA microarrays, gene chip or biochip is a technique in which small DNA fragments are attached to a solid surface. The solid surface is made up of glass, plastic or silicon biochip. The main principle behind microarry is hybridization between attached DNA (DNA) and known sequence DNA (probe DNA). Where the two DNA (probe and target DNA) bind it shows that particular sequence (probe DNA sequence) is present in the target DNA. Thousand of DNA sequence can be detected simultaneously. This technique is routinely used for Cancer’s gene studies. Another example is the use of DNA chips to test drinking water quality. The microbes (present in water)