Biotechnology

Biotechnology applications span agriculture, healthcare, and diagnostics. Three critical research areas of biotechnology: (i) finding the best catalyst (improved organism or pure enzyme); (ii) creating optimal conditions through engineering for the catalyst to act; and (iii) downstream processing technologies to purify the protein/organic compound. --- ### GMOs and Applications in Agriculture **Genetically Modified Organisms (GMOs):** Plants, bacteria, fungi and animals whose genes have been altered by manipulation. GM plants have been useful in many ways: - Made crops more tolerant to abiotic stresses (cold, drought, salt, heat) - Reduced reliance on chemical pesticides (pest-resistant crops) - Helped reduce post-harvest losses - Increased efficiency of mineral usage by plants (prevents early exhaustion of soil fertility) - Enhanced nutritional value of food, e.g., **Vitamin A-enriched rice** (Golden Rice) - Also used to create tailor-made plants to supply alternative resources in the form of starches, fuels and pharmaceuticals **Bt Toxin and Bt Cotton:** - Bt toxin is produced by a bacterium called **Bacillus thuringiensis** (Bt) - Bt toxin gene has been cloned from the bacteria and expressed in plants to provide resistance to insects without need for insecticides (bio-pesticide) - Examples: Bt cotton, Bt corn, rice, tomato, potato and soyabean - Specific **cry genes** encode the proteins. Examples: proteins encoded by **cryIAc** and **cryIIAb** control the cotton bollworms; **cryIAb** controls corn borer - The Bt toxin proteins exist as **inactive protoxins** in the bacteria. Once an insect ingests the inactive toxin, it is converted into an **active form** due to the alkaline pH of the gut which solubilises the crystals. The activated toxin binds to the surface of midgut epithelial cells and creates pores that cause cell swelling and lysis → death of the insect - The toxin does not kill Bacillus because it is inactive (protoxin form) in the bacterial cell **Pest Resistant Plants — RNA Interference (RNAi):** - Nematode **Meloidegyne incognita** infects tobacco plant roots causing great reduction in yield - Strategy: RNAi (RNA interference) — takes place in all eukaryotic organisms as method of cellular defense; involves silencing a specific mRNA by complementary dsRNA - Using Agrobacterium vectors, nematode-specific genes were introduced into host plant producing both sense and anti-sense RNA → complementary to each other → formed dsRNA → initiated RNAi → silenced specific mRNA of nematode → parasite could not survive in transgenic host --- ### Genetically Engineered Insulin - Insulin used for diabetes was earlier extracted from pancreas of slaughtered cattle and pigs; caused allergic reactions in some patients - Insulin consists of two short polypeptide chains: **chain A** and **chain B**, linked together by disulphide bridges. In mammals including humans, insulin is synthesised as **pro-hormone** (pro-insulin) containing an extra stretch called the **C peptide** which is absent in mature insulin - 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 disulphide bonds to form human insulin --- ### Gene Therapy **Gene therapy** is a collection of methods that allows correction of a gene defect diagnosed in a child/embryo. Genes are inserted into a person's cells and tissues to treat a disease. - The **first clinical gene therapy** was given in **1990** to a **4-year-old girl** with **adenosine deaminase (ADA) deficiency**. ADA enzyme is crucial for the immune system to function; disorder caused by deletion of gene for adenosine deaminase - Lymphocytes from the blood of the patient were grown in culture outside the body. A functional ADA cDNA (using a retroviral vector) was introduced into these lymphocytes, which were subsequently returned to the patient - These cells are not immortal → patient requires periodic infusion of such genetically engineered lymphocytes - If the gene is isolated from marrow cells producing ADA is introduced into cells at early embryonic stages, it could be a permanent cure --- ### Molecular Diagnostics Early diagnosis allows effective treatment. Recombinant DNA technology, PCR and ELISA serve the purpose of early diagnosis. - **PCR (Polymerase Chain Reaction):** Amplifies very low concentrations of bacterial or viral DNA (before disease symptoms are visible). Now routinely used to detect HIV in suspected AIDS patients. Also used to detect mutations in genes in suspected cancer patients - **DNA probes:** A single-stranded DNA or RNA tagged with a radioactive molecule (probe) allowed to hybridise to its complementary DNA in a clone of cells; detected by autoradiography - **ELISA (Enzyme Linked Immuno-Sorbent Assay):** Based on principle of antigen-antibody interaction. Detects infection by presence of antigens (proteins, glycoproteins) or by detecting antibodies synthesised against the pathogen --- ### Transgenic Animals Animals that have had their DNA manipulated to possess and express an extra (foreign) gene. Transgenic rats, rabbits, pigs, sheep, cows and fish have been produced; over **95 per cent** of all existing transgenic animals are **mice**. Uses: - Study of normal physiology and development (e.g., genes involved in growth such as insulin-like growth factor) - Study of disease (models for cancer, cystic fibrosis, rheumatoid arthritis, Alzheimer's) --- ### Ethical Issues — Biopiracy and GEAC **Biopiracy** refers to the use of bio-resources by multinational companies and other organisations without proper authorisation from the countries and people concerned and without compensatory payment. **GEAC (Genetic Engineering Approval Committee):** Statutory body in India that makes decisions regarding the validity of GM research and safety of introducing GM organisms for public services. ---

**Biotechnology** deals with techniques of using live organisms or enzymes from organisms to produce products and processes useful to humans. The European Federation of Biotechnology (EFB) definition: "The integration of natural science and organisms, cells, parts thereof, and molecular analogues for products and services." Modern biotechnology rests on two core techniques: 1. **Genetic engineering:** 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 2. **Maintenance of sterile (microbial contamination-free) ambience** in chemical engineering processes to enable growth of only desired microbe/eukaryotic cell in large quantities for manufacture of biotechnological products like antibiotics, vaccines, enzymes, etc. **First recombinant DNA:** **Stanley Cohen and Herbert Boyer** accomplished this in 1972 by linking an antibiotic resistance gene with a native plasmid (autonomously replicating circular extra-chromosomal DNA) of Salmonella typhimurium. This was then transferred into E. coli where it replicated — called **cloning of antibiotic resistance gene in E. coli**. **Three basic steps in genetically modifying an organism:** 1. Identification of DNA with desirable genes 2. Introduction of the identified DNA into the host 3. Maintenance of introduced DNA in the host and transfer of the DNA to its progeny --- ### Restriction Enzymes In 1963, two enzymes responsible for restricting growth of bacteriophage in E. coli were isolated — one added methyl groups to DNA; the other (called **restriction endonuclease**) cut DNA. The first restriction endonuclease **Hind II** was isolated and characterised five years later. It always cuts DNA at a particular point by recognising a specific sequence of six base pairs — the **recognition sequence**. Today we know **more than 900 restriction enzymes** isolated from over 230 strains of bacteria, each recognising different recognition sequences. **Naming convention:** First letter from genus, next two from species of the prokaryotic cell, then Roman numeral for order of isolation. E.g., **EcoRI** from Escherichia coli RY13 (R = strain name, I = first enzyme isolated). **Palindromic sequences:** Each restriction endonuclease recognises a specific palindromic nucleotide sequence. In DNA, a palindrome reads the same on both strands in 5' → 3' direction. Example: EcoRI recognition sequence: - 5' - GAATTC - 3' - 3' - CTTAAG - 5' Restriction enzymes cut strands a little away from the centre of the palindrome sites but between the same two bases on the opposite strands. This leaves single-stranded overhanging stretches called **sticky ends** (named so because they form hydrogen bonds with their complementary cut counterparts). The stickiness of the ends facilitates the action of **DNA ligase**. **Restriction endonucleases** are used in genetic engineering to form 'recombinant' molecules of DNA composed of DNA from different sources/genomes. When cut by the same restriction enzyme, resultant DNA fragments have the same sticky ends and can be joined end-to-end using DNA ligases. --- ### Vectors, Hosts, and the Process **Cloning vectors** carry the foreign DNA into a host organism. A plasmid can be used as a vector to deliver an alien piece of DNA into the host organism. The alien piece of DNA must be linked to the origin of replication so that it can replicate and multiply in the host. **Gel electrophoresis:** Used to separate DNA fragments generated by restriction enzymes based on size. DNA fragments are negatively charged and hence move towards the anode. Separated DNA fragments appear as bands on the gel. Specific DNA fragments can be eluted (extracted) from the gel. **PCR (Polymerase Chain Reaction):** Amplifies specific nucleic acid sequences. Very low concentration of bacteria or virus (before symptoms visible) can be detected by amplification of their nucleic acid by PCR. PCR is routinely used to detect HIV in suspected AIDS patients and mutations in genes in suspected cancer patients. ---