1. Biotechnology Fundamentals
Lecture III-IV
(BIT-110)

2. Biotechnology?
Fundamental Techniques?
Applications?

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

4. 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

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

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

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

8. Biodegradation
Process by which organic substances are broken down by the enzymes produced by living organisms.
The term is often used in relation to ecology, waste management and environmental remediation, bioremediation
Organic material can be degraded aerobically, or anaerobically.

9. Bioremediation
Any process that uses microorganisms, fungi, green plants or their enzymes to return the natural environment altered by contaminants to its original condition.
Bioremediation may be employed to attack specific soil contaminants, such as degradation of chlorinated hydrocarbons by bacteria e.g.
cleanup of oil spills by the addition of nitrate and/or sulfate fertilizers to facilitate the decomposition of crude oil by indigenous or exogenous bacteria.

10. 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 with calcium
This term may also be used to indicate the digestion process in which bacteria utilize the organic part of the matter, leaving behind the minerals

11. Biosurfactants Surface-active substances synthesized by living cells.
They have the properties of reducing surface tension, stabilizing emulsions, 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

12. 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)

13. 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

15. 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

16. 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.

17. 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.

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

19. 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

20. 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.

21. Biogenetic Techniques Gene Splicing
Isolation of DNA molecules,
Cutting of DNA
Restriction Analysis
Rejoining of DNA molecules from different sources
Formation of recombinants
Transformation

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

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

24. 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.

25. 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)

26. 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

27. 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.

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

29. 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

30. 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

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

32. Cloning Technologies Recombinant DNA technology DNA cloning
Molecular cloning
Gene cloning
Reproductive cloning
Therapeutic cloning
Embryo cloning

33. Animal Cloning
Dolly and her surrogate mother.

34. 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.

35. Why Clone Animals? To answer questions of basic biology
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).

36. 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.

37. 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.

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

39. 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

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

41. 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)

42. 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

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

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

45. 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

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

47. 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

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

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

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

52. Thanks