biotechnology chapter of Karnataka PUC

1. BIOTECHNOLOGY
(Karnataka PUC - Biology)
DR. M. JAYAKARA BHANDARY
Associate Professor of Botany
GOVERNMENT COLLEGE, KARWAR

2. Definition
• BT is any technique that uses living organisms
or their parts to make or modify products, to
improve plants or animals, or to develop
micro-organisms for specific uses.
• BT is the application of scientific and
engineering principles to the processing of
materials by biological agents to provide
goods and services

3. BRANCHES & SCOPE OF BT
• Recombinant DNA technology or Genetic Engineering:
Genetically modifying organisms to create new
variety of microbes, plants and animals. Ex.
Insulin producing bacteria, Golden Rice,
Transgenic cattle, etc.
• Tissue Culture: Many plants from small piece of
tissue
• DNA fingerprinting: Identifying criminals, real
parents
• Gene Therapy: Disease treatment by gene
alteration

4. TRANSGENIC PLANTS

5. GENETIC ENGINEERING
• Modifying organisms genetically, by
transferring genes from other organisms using
Recombinant DNA technology
• Creating organisms with new genes –
Genetically modified Organisms (GMO’s) or
transgenic Organisms.
• Addition or Modification of characters

6. TOOLS FOR GE
1. vectors
• DNA molecules that transport foreign genes
into a cell or organism.
• Ex. Plasmids - circular, small, independently
replicating DNA molecules of certain Bacteria
• Artificially Designed –
• Ex. pUC 18 (plasmid designed in University of
California)
• pBR 322 (plasmid designed by Boliver &
Rodrigues)

7. CHROMOSOME PLASMID
PLASMID IN A BACTERIAL CELL

9. 2. RESTRICTION ENZYMES (REN)
• DNA cutting enzymes – DNA scissors
• Cut DNA into double stranded segments at
specific regions called Palindromes ( sequence
of 4-8 base pairs reading the same in both
directions).
• Some produce BLUNT ENDS
• Some produce STAGGERED OR STICKY ENDS

11. 3. DNA LIGASES
• Enzymes that join or ligate the sugar-
phosphate backbones of two DNA fragments
by forming phosphodiester bonds.
• Ex. T4 ligase
• Used to join two DNA pieces cut by REN
(Vector and gene)
• my videosRestrict_enzymes.mov

12. 4. Host cell
• Cells into which the
genes are transferred.
• Bacterial cells are most
suitable because they
are easy to grow,
rapidly.
• Common homultiply st
– Escherichia coli.

13. Eschecell richia coli– Electron micrograph

14. 5. BIOREACTORS
• Large vessels specially
designed to grow
genetically modified
microbes or cells in
large scale and to
harvest the product of
commercial value
• Up to 5,00,000 litres

16. RECOMBINANT DNA TECHNOLOGY
(rDNA technology)
• Recombinant DNA – DNA produced by
combining two or more segments of DNA
taken from different sources.
• Recombinant DNA Technology – technique of
constructing a recombinant DNA
• Important in GE

17. STEPS IN rDNA TECHNOLOGY
1.OBTAINING THE GENE – isolation from cells/from gene libraries/artificial synthesis
2. VECTOR SELECTION - suitable vector selected
3. RESTRICTION DIGESTION – Both gene and vector cut by the same REN to produce sticky ends (Slicing)
4. LIGATION – Gene and vector DNA with sticky ends mixed in the presence of DNA ligase. They join and form
Recombinant DNA (splicing)
5. TRANSFER INTO HOST CELL – Recombinant DNA is inserted into suitable host cell
6. MULTIPLICATION & EXTRACTION OF PRODUCT – Genetically modified cell multiplied (Cloning), product harvested

20. ENJOY AN ANIMATION OF rDNA
TECHNOLOGY
• my videosPLASMIDCLONING.AVI

21. Recombinant Insulin Production

22. STRUCTURE OF INSULIN

23. APPLICATIONS OF rDNA TECHNOLOGY
• Production of GMO’s
• Production of r Proteins – insulin, growth
hormone, Blood clotting factors, etc
• Gene therapy
• Recombinant vaccines

24. DNA FINGERPRINTING
• Technique which helps in the establishment of
identity of a person or organism based on
uniqueness of DNA sequence
• Developed by Alec J. Jeffreys – 1985
• Based on regions of DNA called Variable
Number Tandem Repeats (VNTRs)
• Uses: Identification of criminals, solving
parental disputes, identifying any biological
materials.

25. Steps in DNA fingerprinting
1 . DNA EXTRACTION FROM TISSUE SAMPLES
2. RESTRICTION DIGESTION – cutting DNA into double stranded fragments
3. ELECTROPHORETIC SEPARATION – double stranded DNA fragments separated
according to size by electrophoresis on a gel slab
4. SINGLE STRAND SEPARATION – double stranded DNA separated into single
stands by treating with an alkali
5. SOUTHERN BLOTTING – DNA bands from gel slab transferred to
nitrocellulose sheet
6. PROBE HYBRIDISATION – DNA treated with radioactive DNA probes
7. AUTO RADIOGRAPHY – X-ray photography of nitrocellulose sheet to obtain
DNA fingerprints.
8. COMPARISON OR MATCHING OF DNA FINGERPRINTS

26. • my
videos3ESDSGEL.AVI
• my
videosSOUTHERN.EXE

27. GENE THERAPY
• Treating diseases by manipulating the genetic
material of the patients.
• Addition of healthy gene in the place of
defective gene by recombinant DNA
technology.
• Genetic diseases may be cured
• Viruses are used as vectors

28. TYPES OF GENE THERAPY
Ex vivo gene therapy
• Gene is added to cells separated
from body (outside the body).
• Ex. ADA deficiency/ SCID
In vivo gene therapy
• Gene transferred directly to cells
inside the body.
• Ex. Cystic fibrosis

30. Somatic gene therapy
• Only somatic or body cells are used for
gene therapy.
• Effect short lived, not inherited
Germline gene therapy
• Germ cells (eggs, sperms, zygotes) used
for therapy.
• Effect permanent, inherited. Not
permitted at present for ethical reasons.

31. MONOCLONAL ANTIBODIES
• Antibodies are protein molecules produced by
B-lymphocytes during infection/ antigenic
stimulation.
• Pure antibodies with predetermined
specificity which can react with only a single
type of antigen are called monoclonal
antibodies (MAB’s)
• Polyclonal AB’s react with many different
antigens

32. MAB production
• By HYBRIDOMA TECHNOLOGY of Georges
Kohler & Cesal Milstein, 1975.
• Hybridoma cells – formed by fusion of
antibody forming B lymphocytes and tumour
causing Myeloma cells.
• Two properties – Divide continuously and
produce antibodies.
• Hybridoma cells cultured – MAB’s produced.
• Uses: Blood typing, Diagnostic techniques like
ELISA, etc

34. HUMAN GENOME PROJECT
• Genome – All genes in the haploid set of
chromosomes of an organism.
• Human genome – genes in 24 chromosomes
(22 autosomes + X & Y chromosomes)
• HGP: Project to study the complete sequence
of bases of the human genome
• 1990 – 2003, 3 billion dollars

36. OUTCOME OF HGP
• Total genes in human genome – 30,000
• Total base pairs – 3164.7 million
• 99.9% genes same in all humans
• Only 2% gene s actually code for proteins.
• Function of 98% genes unknown. Junk DNA
• Average size of human genes – 3000 base pairs
• Largest human gene – dystrophin gene- 2.4 million
base pairs.
• Max. genes in Ch. 1 (2968). Smallest in Y ch. (231)

37. Uses of HGP
• Complete understanding of Human genetics
• Discovery of disease related genes , Helps in
developmemt of gene therapy methods. Ex.
Cystic fibrosis, cancers, etc.
• Development of gene diagnosis methods
• my videosLife.mpg

38. IMPROVEMENT OF CROP PLANTS
• BREEDING TECHNIQUES: Production of new
improved crop varieties.
– Hybridisaton – crossing genetically different plants
• Intraspecific, interspecific, intergeneric.
- Mutation breeding
- Selection – Mass selection, Pureline selection, Clonal
selection.
- Polyploidy breeding

39. PLANT TISSUE CULTURE
• Technique of growing isolated plant tissues or
cells in an artificial medium under aseptic
conditions.
• Plant cells TOTIPOTENT. Each cell can Multiply
and develop in to new plants when cultured.
• Growth medium: Nutrients, vitamins,
hormones, agar

40. STEPS IN PLANT TISSUE CULTURE
INOCULATION
TRANSFER TO OPEN
CALLUS FORMATION
HARDENING/ ORGANOGENESIS
ACCLIMATISATION

44. USES OF PLANT TISSUE CULTURE
• Micropropagation
• Disease free plant production
• Multiplication of superior/elite plants
• Somatic hybridisation/cybrid production
• Transgenic plant production
• Secondary metabolite production

45. TRANSGENIC PLANTS
• Genetically modified plants which
contain one or more foreign genes
artificially transferred to their cells.
• Transgenes – Transfection –
transgenesis
• Vector mediated transfer – Ti plasmid
(Agrobacterium tumefaciens), Ri
plasmid (A. rhizogenes)
• Direct gene transfer – micro-injection,
gene gun, electroporation

47. GOLDEN RICE
Transgenic rice produced by Ingo
Potrychus & Peter Bayer – 1999..
3 genes for beta carotene (Vit. A
precursor) production transferred to
rice plant.
Beta carotene produced in endosperm
of rice – golden yellow rice
Prevents Vit. A deficiency problems

48. GOLDEN RICE PRODUCTION
• Isolation of genes: 2 from daffodil plant
( Psy gene – Phytoene synthase, Lyc
gene – lycopene cyclase), 1 from
Erwinia uredovora ( crt 1 gene –
Phytoene desaturase ).
• Insertion of genes to rice embryo cells:
Using Ti plasmid
• Transgenic plant production:
Genetically modified embryos
developed into rice plants.

51. USES OF TRANSGENIC PLANTS
Producing plants with better nutritional
quality. Ex. Golden rice
Producing plants with disease/pest
resistance. Ex. Bt corn
Producing plants with new characters. Ex.
Vaccine producing plants.

52. IMPROVEMENT OF ANIMALS
Mating: Crossing two selected parent animals
to produce superior progeny
Artificial Insemination: Semen (sperms) of
superior males artificially placed in the
reproductive tract of female animals to effect
fertilisation. More females can be fertlised by
superior males. Ex. Cattles, horses.
Multiple Ovulation and Embryo transfer
(MOET): Superior females induced to produce
more eggs per cycle, Fertilised to form many
embryos, Embryos collected and transplanted
to surrogate mothers. Useful to multiply
superior females.

53. STEM CELL CULTURE
• Immature cells with the potential to
develop into many different cells of
animal body.
• PLURIPOTENT/MULTIPOTENT
• Adult stem cells & embryonal stem
cells.
• Can be cultured to form muscle cells,
nerve cells, etc for transplantation
therapy.
• Cultured stem cells can also be used
for genetic modification (Bone marrow
cells).

56. TRANSGENIC ANIMALS
• Genetically modified Animals with
one or more foreign genes.
• Gene transfer by viral vectors &
Micro-injection method.
• Genes introduced to eggs or
early embryonal cells, induced to
form zygotes, transplanted into
surrogate mothers.

57. TRANSGENIC CATTLE PRODUCTION
(Nuclear transfer technology)

58. USES OF TRANSGENIC ANIMALS
• Production of recombinant
proteins . Ex. Human growth
hormone, Clotting factor.
• Models to study human diseases:
Oncomouse,
• Production of improved animals:
Fast growing pig, cattle, etc.

59. HAZARDS & SAFEGUARDS OF GE
Dangerous GE research strictly
organisms – supervised.
pathogens – Bio Permission of rDNA
terrorism. advisory committee
Food from GMO’s essential.
dangerous to health GM organisms
GM crops also kill cannot be released
useful insects – to open without
butterflies proper testing.
Modifying human Labelling GM foods
germ cells – against compulsory.
nature, unethical Human GE banned.