Biotechnology Fundamentals

Biotechnology Fundamentals Lecture III-IV (BIT-110)

Biotechnology? • Fundamental Techniques? • Applications?

Fermentation Animal cloning Genetically modified foods and the American-European opinion divide. Gene cloning for pharmaceutical production DNA fingerprinting The promise and perhaps perils of embryonic stem cells Biotechnology

1. Fermentation • A technique that requires bioreactors to allow a biological process under controlled (optimum) conditions producing a useful substance in large amount • Alcoholic drinks • Production of wine and beer • Recently mammalian cells

Can grow trillions of new bacteria One cell with the recombinant plasmid A fermentor used to grow recombinant bacteria.

2. Biotransformation • chemical modifications made by an organism on a chemical compound. • Microbial Biotransformation • Biodegradation • Bioremediation • Mineralization • Biosurfactant

Microbial biotransformation • Biotransformation of variouspollutantsto clean up contaminated environments • The methods harness the naturally occurring, microbial catabolic diversity to degrade, transform or accumulate a huge range of compounds includinghydrocarbons (e.g. • oil, • PCBs (polychlorinated biphenyl), • PAHs (polyaromatic hydrocarbons), • pharmaceutical substances,and • metals. • Biological processes play a major role in the removal ofcontaminantsandpollutantsfrom theenvironment.

Biodegradation • Process by whichorganicsubstances arebroken downby theenzymesproduced by living organisms. • The term is often used in relation toecology,waste managementandenvironmentalremediation, bioremediation • Organic material can be degradedaerobically, oranaerobically.

Bioremediation • Any process that usesmicroorganisms,fungi,green plantsor theirenzymesto return the natural environment altered by contaminants to its original condition. • Bioremediation may be employed to attack specificsoil contaminants, such as degradation ofchlorinatedhydrocarbonsbybacteria e.g. • cleanup ofoil spillsby the addition ofnitrateand/orsulfate fertilizersto facilitate the decomposition ofcrude oilby indigenous or exogenous bacteria.

Mineralization • The process where a substance is converted from an organic substance to an inorganic substance. • modification ends in mineral compounds like (NH+3 or H2O), • This may also be a normal biological process which takes place during the life of an organism such as the formation of bone tissue or egg shells, largely withcalcium • This term may also be used to indicate thedigestionprocess in which bacteria utilize the organic part of the matter, leaving behind the minerals

Biosurfactants • Surface-active substances synthesized by living cells. • They have the properties of reducingsurface tension,stabilizingemulsions, promoting foaming and are generally non-toxic and biodegradable. • Biosurfactant producing microorganisms may play an important role in the accelerated bioremediation of hydrocarbon contaminated sites. • These compounds can also be used in enhanced oil recovery and may be considered for other potential applications in environmental protection

3. Cell fusion • Involves combining two cells to make a single cell that contains all the genetic material of the original cells. • Used to create new plants by fusing cells from species that do not hybridize naturally • Crossbreeding and then generation of new plant from the fused cell e.g. POMATO (fusion of tomato and potato) • Used to generate monoclonal antibodies • (Protective proteins produced by a clone of a single cells)

How cells can fuse? • Using Viruses • Using chemicals (polymers) • Weaken the cell wall of the cells and cause them to bind together • Using Enzymes • Plant cells can be fused only after removing the cell walls completely by enzymatic reactions

4. Liposomes • Microscopic spherical particles that are formed when lipids form a suspension in water. • Lipid molecules are arranged in a manner so as to enclose a tiny space in the centre to carry a drug or any other material • Used as vehicles for delivering certain drugs to the target tissues of the body • Drugs are encapsulated in liposomes • protect against digestive enzymes in the stomach

The spontaneous closure of a phospholipid bilayer to form a sealed compartment. Closed structure is stable because it avoids the exposure of the hydrophobic hydrocarbon tails to water (energetically unfavorable.

PHOSPHOLIPID BILAYERS A bilayer of phospholipids forms a sphere in which water is trapped inside. The hydrophilic phosphate regions interact with the water inside and outside of the sphere. The fatty acids of the phospholipids interact and form a hydrophobic center of the bilayer.

Liposomes (A) An electron micrograph of unfixed, unstained phospholipid vesicles. (B) Schematic representation of lipososmes

5. Cell and tissue culture • Growth of living cells or organism outside the body in a suitable culture medium which provides nutrients to the growing cells. • Individual cells grow and divide in s sterile medium. • Extensively used in Labs e.g. • Cancer Research • Plant breeding • Karyotyping (chromosomal analysis) • Organized profile of a person’s chromosomes

6. Genetic Engineering • The transfer of genes (segment of DNA) from one species to another. • Requires recombinant DNA technology • Gene transfer techniques • Used to improve breeds of economically important plants, cow, gorses, dogs, etc.

Biogenetic TechniquesGene Splicing • Isolation of DNA molecules, • Cutting of DNA • Restriction Analysis • Rejoining of DNA molecules from different sources • Formation of recombinants • Transformation

Transformation? • Introduction of foreign DNA into the cells (Prokaryotic cells, bacteria) using non-viral methods • Conversion of normal cell into cancerous cell

Transfection? • Transfection is the process of introducing nucleic acids into cells (eukaryotic cells such as fungi, algae and plants) by non-viral methods.

Transduction? • Naturally, process by which DNA is transferred from one bacterium to another by a virus. • In biotechnology, it refers to the process whereby foreign DNA is introduced into another cell via a viral vector.

Gene Therapy • Insertion of normal gene from one organism into the patient’s cells (where gene is defective) • Post-transcriptional gene silencing using small pieces of RNA (RNAi)

DNA Fingerprinting (DNA typing, DNA testing etc.) • To identify various components of DNA, unique for an individual • A portion of the DNA of an organism uniquely distinguishes that organism from another • Technique is based on mini-satellites, repeated several times in the genome

DNA Footprinting • Detects DNA-protein interaction using the fact that a protein bound to DNA will protect that DNA from enzymatic cleavage. • Using this technique, it is possible to locate a protein binding site on a particular DNA molecule.

Gene Mapping • Mapping of genes to specific location on chromosome • Assigning DNA fragments to chromosomes

In vitro;within the glass, outside the living system • Performing a given procedure in a controlled environment outside of a living organism • In vivo;within the living • Experimentation using a whole, living organism as opposed to a partial or dead organism • In situ; in the place (usually means something intermediate between in vivo and in vitro). • Examining a cell within a whole organ intact • Ex vivo;out of the living • experimentation or measurements done in or on living tissue in an artificial environment outside the organism with the minimum alteration of the natural conditions

Cloning • A technique used for production of organism from a single individual, which are identical in genetic constitution. • A clone is an organism that is derived from a single parent through non-sexual methods. • Plants • Microorganisms • Not in mammals • Cloning of mammals (Nuclear Transfer) • as practiced in Dolly at Roslin Institute of Edinburgh • Dolly was unable to breed and produce healthy offsprings

Embryo Transfer • To improve dairy cattle • Artificial insemination • Surgical removal of a fertilized egg • Transfer into a recipient female

Cloning Technologies • Recombinant DNA technology • DNA cloning • Molecular cloning • Gene cloning • Reproductive cloning • Therapeutic cloning • Embryo cloning

Animal Cloning Dolly and her surrogate mother.

The Biotechnology of Reproductive Cloning Even under the best of circumstances, the current technology of cloning is very inefficient. Cloning provides the most direct demonstration that all cells of an individual share a common genetic blueprint.

Why Clone Animals? Five genetically identical cloned pigs. To answer questions of basic biology For pharmaceutical production. For herd improvement. To satisfy our desires (e.g. pet cloning).

Is Animal Cloning Ethical? The first cloned horse and her surrogate mother/genetic twin. As with many important questions, the answer is beyond the scope of science.

Saved by Cloning? Some are firm believers while many view these approaches to be more of a stunt. Note the use of a closely related species, a domestic goat, as egg donor and surrogate mother.

(Science (2002) 295:1443) Carbon Copy– the First Cloned Pet Significantly, Carbon Copy is not a phenotypic carbon copy of the animal she was cloned from.

DNA Cloning • Transfer of a DNA fragment of interest from one organism to a self-replicating genetic element such as a bacterial plasmid • Plasmids • Self-replicating extra-chromosomal circular DNA molecules, distinct from normal bacterial genome

DNA Cloning - Uses • Gene therapy • Genetic engineering of organisms • Genome sequencing

Reproductive Cloning • A technology used to generate an animal that has same nuclear DNA as another currently or previously existing animal • Dolly • How Is Reproductive Cloning Done? • Somatic cell nuclear transfer (SCNT)

Therapeutic Cloning • Production of human embryos for use in research • Goal • To harvest stem cells that can be used to study human development and to treat disease

Genetically Modiﬁed Cloned Offspring • Biotechnological applications • Production of pharmaceuticals • Xenotransplantation • Study and eradication of human disease • Improvement of livestock

Production of Pharmaceuticals • Insulin for diabetes • Interferon for viral infections • Tissue plasminogen activator (which dissolves blood clots)

Xenotransplantation • To develop animals whose organs will not cause an immunological response and destroy transplanted tissue when transferred to humans • Pig organs • Hearts, lungs, kidneys, liver • Neural tissue for Parkinson's • Islets cells for diabetes patients

Study Human Disease • Sheep model to investigate human cystic ﬁbrosis • Cloned sheep used for drug testing and to evaluate new therapies

Improvement Of Livestock • Cloning animal with excellent traits • Production of a large number of clones from high quality animals • Allow overall genetic improvement of herd • Repopulate endangered animals

Animal Cloning • Sheep, cattle, goats, pigs, and mice • Cloning efforts in rabbits, rats, cats, dogs, and horses are ongoing

Risks Of Cloning • Reproductive cloning expensive and highly inefficient • > 90% of cloning attempts fail to produce viable offspring

The Next Step? Highly unlikely Attempts at human cloning are viewed very unfavorably in the scientific community

Biotechnology Fundamentals