Pharmaceutical Biotechnology
ABOUT THE BOOK: One should respect the power of biotechnology and
apply it for the benefit of humankind.
Biotechnology in pharmaceutical industry employs techniques of modern molecular
biology to help humankind. Pharmaceutical biotechnology works to make
manufacturing processes more efficient in industrial production of
pharmaceutical products. Biotechnology will transform the industrial sector in
much the same way that it has changed the pharmaceutical sector.
Pharmaceutical Industrial Biotechnology companies develop biocatalysts, such as
enzymes, to be used in chemical synthesis. Enzymes are proteins produced by all
living organisms. In humans, enzymes help digest food, signal cells to turn on
and off, and perform other complex functions. Enzymes are characterized
according to the compounds they act upon. Some of the most common enzymes are
proteases, which break down protein; celluloses, which break down cellulose;
lipases, which act on fatty acids and oils; and oils; and amylases, which break
starch down into simple sugars. To improve the productivity-to-cost ratio,
scientists are modifying genes to increase enzyme productivity in microorganisms
commonly used in manufacturing. They also use genetic engineering to make other
microbial enzymes that are too expensive or even impossible to cultivate. The
biotechnology techniques of protein engineering and directed protein evolution
have been used to modify the substrate specificity of enzymes, improve catalytic
properties or broaden the reaction conditions under which enzymes can function
so that they are more compatible with existing industrial processes. Companies
involved in industrial biotechnology are constantly striving to discover and
develop high-value enzymes and bioactive compounds that will enhance Current
industrial processes.
The present book is an attempt to provide overall information to the readers
interested in Pharmaceutical Biotechnology.
AUTHOR: S. S. Purohit, H. N. Kakrani & A. K. Saluja | ISBN: 9788188826025 | YEAR: 2010 | PAGES: 528 | SIZE: 18.5 x 24 x 2.5 cm. | BINDING: Soft | LANGUAGE: English
CONTENTS:
1. PHARMACEUTICAL BIOTECHNOLOGY: AN INTRODUCTION
a) Origin and Definition: Old Vs New
i. Biotechnology
ii. Definitions
iii. Historical Background
b) Scope and Importance of Biotechnology
c) Genetic Engineering
d) Tissue Culture
e) Protoplast Culture and
i. Micropropagations
f) Biosensors, Biochips, Biofilms and Biosurfactents
g) The Biotechnologies and their applications
i. Monoclonal Antibody Technologies
ii. Cell Culture Technology
iii. Cloning Technology
iv. Genetic Modification Technology
v. Protein Engineering Technology
vi. Hybrid Technologies
vii. Biosensor Technology
viii. Tissue Engineering Technology
ix. DNA Chip Technology
x. Bioinformatics Technology
h) Research Promotion and Priorities in India
i. Bioinformatics
ii. Plant Molecular Biological
Researches
iii. Service Oriented Infrastructural
Facilities
i) Biotechnology Industry Statistics: Some Facts
2. BIOLOGICAL GENETICS AND VARIATIONS IN CHARACTERS OF MICROBES
a) Importance of Microbial Genetics
i. Important Terms Used in Microbial
Genetics
b) DNA as Genetic Material
c) DNA Replication
i. How replication is achieved?
ii. How Bacteria Replicate Their DNA
d) Ribonucleic Acid (RNA)
e) Gene
i. Prokaryotic Genes
ii. Structure of Cyanobacterial Genes
iii. Gene Arrangement in the
Cauliflower Mosaic Virus
iv. Eukaryotic Genes
v. Organelle Genes
vi. Gene That Code for Proteins
vii. Gene That Code for tRNA and tRNA
viii. Eukaryotic Gene Organization
and Expression
ix. Prokaryotic Gene Organization and
Expression
f) Genetic Code
i. Concepts of Degeneracy
ii. DNA Encodes Genetic Information
g) Molecular Cloning of DNA
i. Restriction Enzymes
ii. DNA Fingerprinting
iii. Plasmids
h) Cell Properties Determined by Plasmid Genes
i) Genome
i. Genomes of Microorganisms
ii. Genomes of Eukaryotic
1.
Microorganisms
iii. Genomes of Prokaryotic
Microorganisms
iv. Importance of Genome in Research
j) Mapping Genomes by Genetic Techniques
i. Genetic and Physical Maps
ii. Markers for Genetic Maps
iii. Simple sequence length
polymorphisms (SSLPs)
iv. Southern hybridization
v. DNA chip and Its Technology
3. CONCEPTS, PERSPECTIVES AND TECHNIQUES OF MICROBIAL GENETIC AND PROTEIN
ENGINEERING
a) Advantages of Genetic Engineering
i. Practical Applications
b) Important Terms of Genetic Engineering
c) Prerequisites in Plant Genetic Engineering Methods
d) Perspectives of Genetic Engineering
i. Other Applications of Genetic
Engineering
e) Protein Engineering: Trends and Basic Concepts
i. Enzyme Characteristics as Target
for Protein Engineering
ii. Single Cell Protein (SCP)
Production
iii. Protein Engineering:
Achievements
f) Techniques of Genetic Engineering
i. Cleaving DNA With Restriction
Enzymes
ii. Gel Electrophoresis
g) Blotting Techniques
i. Analysis of DNA by Southern
Blotting
ii. Analysis of RNAs by Northern Blot
Hybridizations
iii. Analysis of Proteins by Western
Blot Techniques
h) Detection of RFLPs
i) DNA Sequencing
i. Methods for Analyzing DNA Sequence
ii. Sequencing by Chemical
Degradation
iii. Sequencing by Chain Termination
iv. Direct DNA sequencing using PCR
(also called ligation mediated PCR = LMPCR)
v. Automated DNA Sequence Analysis
j) Chemical Synthesis of DNA
i. Chemical synthesis of tRNA Genes
k) Insertion DNA Sequencing
i. Synthesis of Gene for Yeast Alanyl
tRNA
ii. Synthesis of Gene for a True
Precursor tRNA
iii. Total Synthesis of a Human
Leukocyte Interferon Gene
l) Gene Synthesis Machines
m) Other Techniques of Genetic Engineering
i. Transposable Elements in
Prokaryotes
n) Agroinfection
i. Biolistic Plant Transformation
Microinjection-Mediated Gene Delivery
ii. Micro laser
iii. Electroporation,
4. TECHNIQUES OF RECOMBINANT DNA TECHNOLOGY FOR PRODUCTION OF BIOLOGICALS
a) Recombinant protein production
i. Bio-activity of Expressed Protein
ii. Synthesis and Secretion of
Recombinant Proteins
iii. Xenotransplantation
b) Production of Biologicals
i. Production of Human Insulin
ii. Production of Human Growth
Hormone
iii. Production of Growth Hormone
Releasing Factor (GRF)
c) Production of Erythropoietins (EPO)
d) Interferons (IFN)
e) Interferon or Antiviral Substances
f) Tumour Necrosis Factor (TNF) and Lymphotoxin (LT)
g) Interleukines (IL)
h) Other Products
i) Vaccines
i. Sub-unit Vaccines
ii. Killed Vaccines
iii. Live Vaccine
j) Processes for Recovery and
i. Purification of Biologicals
5. ANIMAL CELL AND TISSUE CULTURE
a) Advantages and Disadvantages of Tissue Culture Methods
b) Primary and Established Cell Lines
c) Kinetics of Cell Growth
i. Established Cell Lines
ii. Primary Cell Lines
iii. Interaction Among Cells
d) Genetics of Cultured Cells
i. Metabolism
e) Animal Cell and Tissue Culture
i. Mechanical Technique
ii. Biochemical Technique
f) Animal Tissue Culture Media
i. Culture Media Containing Naturally
Occurring
1.
Ingredients
ii. Complex Natural Media
g) Laboratory Facilities for Tissue Culture
h) The Substrate on Which Cells Grow in Laboratory
i. Treatment of Substrate Surfaces
ii. Feeder Layers on Substrate
iii. The Gas Phase for Tissue Culture
i) Culture Procedures
i. Preparation and Sterilization of
Glassware and Apparatus
ii. Preparation and Sterilization of
Reagents and Media
iii. Preparation of Animal Material
j) Primary Culture, Cell Lines and Cloning
i. Disaggregation of Tissue and
Primary Culture
ii. Isolation of Tissue
iii. Enzymatic Disaggregation
iv. Disaggregation by trypsin (for
embryonic tissue) or Trypsinization
v. Disaggregation by Collagenase (for
embryonic, normal and malignant tissues)
vi. Mechanical Disaggregation
vii. Separation of Viable and
Nonviable Cells
k) Somatic Cell Fusion
l) Tissue Cultures
i. Primary Explanation Techniques
ii. Slide or Coverslip Cultures
iii. Flask Cultures
iv. Test Tube Cultures
m) Organ Culture
i. Culture of Embryonic Organs
ii. Organ Cultures on Plasma Clots
iii. Organ Cultures on Agar
n) Whole Embryo Culture
i. Culture of Chick Embryo
(Spratt,1956)
o) Animal Tissue Cultures in Biomdical Research-Genetics
i. Karyological Studies
ii. Identification and Study of
Hereditary Metabolic
1. Disorders
iii. Somatic Cell Genetics
iv. Tissue Cultures in Biomedical
1.
Research-Virology and Host parasite Relationships
v. Tissue Cultures in Biomedical
Research-Cancer
6. HYBRIDOMAS TECHNOLOGY AND MONOCLONAL ANTIBODIES
a) Hybridoma
i. Monoclonal B-cells
ii. Types ofMAbs
iii. Chimeric MAbs
b) Somatic Cell Fusion
c) Hybridoma Technology
d) Production of Monoclonal Antibodies
i. Step I: Fusion and Culture of
Hybridomas
ii. Step II: Cloning and Preservation
of Hybridomas
iii. Step III: Production of Antibody
e) Application of Monoclonal Antibodies
7. GENE THERAPY
a) Gene-transfer system
i. Viral vector
ii. Non-viral
b) Physical
c) Viral vectors
i. Adenovirus
ii. Retrovirus
iii. Adeno-associated Virus
d) Non-viral approach
i. Naked DNA
ii. Chemical Methods
e) Physical methods
i. Needle-free injection
ii. Electroporation
iii. Protection and intracellular
release of DNA
f) Recent development and future prospects
8. MICROBES IN PHARMACEUTICAL INDUSTRY
a) Single Cell Protein
i. Microorganisms and their
Fermentation Substrates
ii. Environmental Conditions for
Growth
iii. Choice of Fermentable Materials
iv. SCP Products
v. Harmful Effects of SCP Use
b) Alcoholic Fermentation
c) Microbes in Organic Acid Production
d) Microbes in Enzyme Production
e) Microbes in Medicine
i. Antibiotics
ii. Ergotin
iii. Vitamins
iv. Glycerin
f) Microbes as Tools for Biological Research
i. Genetical, Cytological and
Biochemical Studies
9. DEVELOPMENT OF INDUSTRIAL MICROORGANISMS
a) Mutation
i. Mutant Detection
ii. Mutant Selection
b) The selection of mutants producing high yield of primary
metabolites
c) The Selection of Secondary Metabolite Producing Mutants
10. CULTURING OF INDUSTRIAL MICROORGANISMS
a) Inoculum Development
b) Batch Culture
c) Continuous Culture
d) The kinetics of Continuous Culture
e) Fed Batch Culture
f) Culture media
g) Precursors and Inducers
h) Repressors
11. INDUSTRIAL FERMENTERS
i. Continuous Culture
a). The components of a fermentor
b). Parts of fermentors
i. Vessel
c) Peripheral parts and accessories
i. Reagent pumps
ii. Medium feed pumps and reservoir
bottles
iii. Rotameter/gas supply/
iv. Sampling device
d) Alternative vessel designs
i. Air lift
ii. Fluidized bed
iii. Hollow fibre
iv. In situ sterilizable fermentors
v. Containment
e) Additional accessories and peripherals
i. Feed pumps
ii. Exit gas analysis
f) Types of reactors
g) Mechanically agitated stirred tank reactors
i. Air-lift Bioreactor and Modified
Air Lift Bioreactors
ii. Novel Seesaw Bioreactor
h) Different types of fermentor instrumentation
i) Analogue controllers – rack system
j) Analogue controllers – separate modules in housings
k) Digital controllers – embedded microprocessor
l) Digital controllers – process controllers
m) Digital controllers – direct computer control
n) Common measurement and control systems
i. Speed control
ii. Temperature control
iii. Control of gas supply
iv. Control of pH
v. Control of dissolved oxygen
vi. Antifoam control
o) Simple continuous culture
p) Fermentor preparation and use
q) Scaling-up of Fermentation
i. Disassembly of the vessel
ii. Cleaning
iii. Preparations for autoclaving
iv. Autoclaving
v. Set-up following autoclaving
vi. Sampling from a fermentor vessel
vii. Inoculation of a fermentor
vessel
r) Maintenance of Aseptic Conditions
s) Transfer of Oxygen
12. PRODUCTION OF ANTIBIOTICS
a) Screening of Antibiotic Producers
b) β- Lactam Antibiotics
i. Penicillins
ii. Cephalosporins
c) Aminoglycoside Antibiotic
i. Streptomycin
d) Tetracyclines
i. Production of Tetracycline
e) Chloramphenicol
i.
Chloramphenicol Production
f) Griseofulvin
i. Inoculum
ii. Medium
iii. Production
iv. Extraction and Purification
g) Macrolide Antibiotics
i. Erythromycin
h) Rifamycins
i. Inoculum
ii. Medium
iii. Fermentation
iv. Recovery and Purification
13. PRODUCTION OF VITAMINS
a) Vitamins: General Aspects
b) Vitamin B Complex
i. Vitamin B12 (Cobamide) Production
c) Vitamin B2, (Riboflavin) Production Production by
Fermentation of Ashbya gossypii (NRRL Y, 1056)
d) Vitamin C (Ascorbic Acid)
14. PRODUCTION OF ERGOT ALKALOIDS
a) Producing Organisms
i. Genetic Considerations
ii. Physiology of Alkaloid Formation
iii. Ergot Alkaloid Enzymes and other
factors
b) Commercial Production in Bioreactors
i. Medium
c) Preparation of Inoculum
d) Production of Alkaloid
i. Peptide Alkaloids
e) Isolation Separation and Purification
i. Peptide Alkaloids
ii. Paspalic Acid
15. OTHER PRIMARY METABOLITES OF PHARMACEUTICAL USES:
a) Fermentation Technology
b) Industrial Alcohol
c) Production of Alcoholic Beverages
d) Amino Acids
e) Glutamic Acid
f) L-Lysine
g) Citric Acid
h) Lactic Acid
i) Clinical Dextran
16. BIOTRANSFORMATION AND STEROID PRODUCTION
a) BioTransformation
b) Methods Used in Biotransformation Process
i. Steroid Bioconversion Fermentation
ii. Analysis and Isolation of Product
iii. Selection of Organisms
c) Biotransformation Process with Special Reference to
Steroids
i. Hydroxylation
ii. Dehydrogenation
iii. Hydrogenation
iv. Epoxidation
v. Aromatization of Ring A
vi. Synthetic Routes
17. BIOLOGY OF ENZYMES
a) Enzymes: the Tailor's Scissors
b) Properties of Enzymes
c) Enzymes: Nomenclature
d) Enzyme Classification
e) Enzymes as Chemo-thermo Regulators
f) What Enzymes Do?
g) What is Enzyme Kinetics?
h) How Enzymes Work
i) Enzyme Activity
i. Multi-substrate Enzymes
j) Enzyme Optima
k) Enzyme Inhibitors, Activators and Inactivators of Enzymes
i. Reversible and Irreversible
Inhibition
l) Enzyme Secretion by Plant Cells
i. The Mechanism of Protein Secretion
ii. The Control of Secretion
m) Sources of Enzymes
n) Microbial Enzymes
o) Mechanism of Enzyme Biosynthesis in Microbial Cells
i. Induction
ii. Repression
iii. Catabolic repression
iv. Transcription control during the
growth cycle
p) Genetics of Enzyme Formation
q) Isoenzymes
i. The Cause of Enzyme Multiplicity
r) Primary or Genetic Isoenzymes
s) Isoenzymes due to Multiple Genetic Loci
t) Secondary or Post-translational Isoenzymes
18. PRODUCTION OF ENZYMES
a) Solid Substrate Fermentation
i. Fermenters
ii. Medium
iii. Advantages
iv. Disadvantages
b) Submerged Fermentation
i. Steps of Enzyme Production
ii. Factors Affecting Submerged
Culture
c) Extraction of Enzyme
i. Physical Disruption Method
ii. Chemical Treatment Method
d) Purification of Enzyme
e) Removal of Nucleic Acids and Cell Debris
f) Preliminary Purification
g) Final Purification
19. PRODUCTION OF ENZYMES BY FERMENTATION
a) Oxidoreductases
i. Pyridine Nucleotide Dehydrogenases
ii. NAD/NADP Dehydrogenase
b) Oxidases
i. Production of Glucose Oxidase
ii. Cholesterol Oxidase
c) Hydrolases
i. Glycosidases
ii. Cellulose
iii. Proteolytic Enzymes
d) Penicillin Amidases (penicillin acylase)
e) Transferases
i. Glycosyl Transferases
ii. Cyclodextrin glycosyl transferase
f) Glycerol Kinase
g) Glucose Isomerase
i. Production of Glucose Isomerase
using Bacillus coagulans
ii. Production by Streptomyces
wedmorensis
20. APPLICATIONS OF ENZYMES
a) Applications in Pharmaceutical Industries
i. Synthesis of Amino Acids
ii. Synthesis of Nucleosides and
Nucleotides
iii. Synthesis of Other Compounds
b) Applications in Therapeutics
i. For Treatment of Gaucher's,
Tay-sach 's, and Fabry's Diseases
c) Clinical Analysis
i. Transferases
d) Ornithine transcarbainylose
e) Creatine kinase
f) Oxidoreductases or Dehydrogenases
g) Hydrolases
21. ENZYME IMMOBILIZATION
a) Important Features Enzyme Immobilization
b) Disadvantages of Enzymes
c) Advantages of immobilized Enzymes
d) Properties of Immobilized Enzymes
i. Stability
ii. Kinetic Properties
e) Types/Methods of Immobilization
f) Choice of Immobilization Method
g) Comparison and evaluation of Immobilization Techniques
h) Effects of Immobilization on Kinetics and Properties of
i. Enzyme
ii. Michaelis Constant
iii. Maximum Reaction Velocity (Vmax)
i) Immobilization Techniques for Cells
i. Cross-linking
ii. Copolymerization
iii. Covalent Binding
iv. Adsorption
v. Entrapment
j) Bioreactors
i. Fennenter Design Considerations
ii. Immobilized plant cell fermenters
iii. Design and Applications of
Bioreactors
k) Choice of Enzyme Reactor
22. APPLICATIONS OF IMMOBILIZED ENZYMES
a) Pharmaceutical Applications
i. Uses in Antibiotics Production
ii. Steroids Production
iii. Amino Acids Production
iv. Acids Production
v. Other Organic Compounds Production
b) Applications in Clinical Analysis
i. Enzyme Electrodes (Biosensors)
ii. Biosensor for Analysis of Organic
Compounds
iii. Conventional Biosensor
iv. Microbial Biosensor
v. Development of Micro biosensor
vi. Biosensors Using Amorphous
Silicon ISFET
vii. Micro-Oxygen Electrode
viii. Integrated Multi-biosensor
ix. Novel Biosensor Based on New
Transducers
23. PLANT TISSUE CULTURE: BASIC CONCEPTS
a) Importance of Tissue Culture
i. Global Picture of Plant Tissue
Culture
b) Plant Tissue Culture: Principles
i. Callus Culture
ii. Meristem Culture
iii. Organ Culture
c) The Concept of Totipotency of Cells
24. PLANT TISSUE CULTURE: METHODS
a) Media preparation: Area/Room
i. Requirements
ii. Z-Cabinets or Shelves
iii. Culture Media, Washing Powder/
Liquid Detergent,
1.
Disinfectants
b) Other Requirements
c) Aseptic Transfer Chamber Area
d) Environmentally Controlled Culture Room
i. Analytical Room
ii. Acclimatization Room
iii. Miscellaneous items
iv. Specifications of Laboratory
Equipments
e) Aseptic Technique
f) Sterilization of Plant Tissues
i. Cleaning (Preparation of
Glasswares/Plastic wares (Autoclavable)
ii. Sterilization
iii. Surfactants
g) In Vitro Environment
h) Pretreatment to Explant Tissues Prior to Culture
i. Procedure
i) Micropropagation through Organogenesis
j) Culture Media and Preparation
k) Media Components
i. Inorganic Salts
ii. Plant Growth Regulators
iii. Carbon Source
iv. Gelling Agent
v. Amino Acids and Amides
vi. Antibiotics
vii. Natural Complexes
l) Additional Requirements
i. Trypticase-Soy Broth Medium
ii. Sterilization of Media
iii. Use and Storage of Coconut Water
m) Related Procedures
i. Ultraviolet Light
ii. Preparation of Phenol
iii. Working with 32P Labelled
Compounds
iv. Silanization of Plastic and
Glassware
25. PROTOPLAST FUSION TECHNOLOGY
a) Importance of Protoplast Isolation and Culture
b) Isolation of Protoplast from Various Plant Parts
i. Enzymatic method
ii. Mechanical method
c) Enzymic Isolation of Protoplasts: Method
d) Methods of Protoplasts Culture
i. Laboratory Facilities
ii. Enzyme Mixture and Osmotic
Stabilizer (Osmoticaj
e) Purification of Isolated Protoplasts
i. Sedimentation and Washing
ii. Flotation
iii. Other Purification Methods
f) Protoplast Viability Testing
i. General Steps of Protoplast
Culture
g) Culture Media for Protoplast Culture
i. Protoplast Culture Media for PC I
Group
ii. Protoplast Culture Media PC II
Group
iii. Protoplast Culture Media PC III
Group
iv. Agar Embedded Culture
v. Microchambers
vi. Hanging Drop Cultures (HDC)
Techniques
vii. Multidrop Array (MDA) Techniques
h) Growth and Division of Protoplast
i. Protoplast Culture: Regeneration
of Cell Wall
ii. Development of Callus/Whole Plant
i) Handling of Regenerated Plantlets
j) Protoplast Fusion
i. General Steps of Protoplast Fusion
k) Protoplast Culture: Regeneration of Plants
i. Organogenesis
ii. Embryogenesis
iii. Handling of Regenerated
Plantlets
l) Protoplast Culture: Uptake of Foreign Materials
i. Incorporation of Foreign DNA
ii. Incorporation of Nuclei
iii. Incorporation of Chloroplasts
iv. Incorporation of Cyanobacteria
v. Incorporation of Bacteria
vi. Incorporation of Virus
m) Incorporation of Non-biological Materials
n) Selection of Fusion Hybrids
i. Visual Selection
ii. Fluorescent Labels
iii. Fluorescence Activated Cells
Sorting
iv. Nutritional Selection
v. Drug Sensitivity and Resistance
o) Somatic Cell Hybridization or Cybrid or Cytoplast
p) Protoplasts for Isolation of Cell Components
26. DNA ISOLATION AND SEQUENCING
i. Isolation of total cellular
DNA
ii. Nucleic Acid Hybridization
a). Methods for Labelling Nucleic Acids
b) Methods of Labelling Nucleic Acid and Probes
i. Nick translation
ii. Primer Extension Method
iii. Methods based on RNA polymerases
c) End-Labelling of Nucleic Acids
d) Choice of Label
e) Mapping Genes on Specific Chromosomes
i. In-situ Hybridization
ii. Transposon Tagging
iii. Genetic Linkage Mapping
f) Genomic Library:
g) Cleaving DNA With Restriction Enzymes
i. Gel electrophoresis
h) Blotting Techniques
i. Analysis of DNA by Southern
Blotting
ii. Analysis of RNAs by Northern Blot
Hybridizationsi
iii. Analysis of Proteins by Western
Blot Techniques
i) Detection of RFLPs'A
j) DNA Sequencing
i. Methods for analyzing DNA sequence
k) Sequencing by chemical degradation
l) Sequencing by chain termination
m) Automated DNA sequence analysis
n) Mechanical Shearing of DNA
o) Transcript Mapping Techniques
i. Primer extension
ii. S1 mapping
p) Synthesis of cDNA from mRNA
i. Preparation of Double-Stranded
cDNA
ii. cDNA Library
q) Searching for Gene Using Computers
27. GENE CLONING VECTOR/PLASMID
a) Plasmids
i. Replication of Plasmids
ii. Size of Plasmids
iii. Copy Number
iv. Amplification of the Plasmid
v. Types of Plasmids
vi. Isolation of Plasmid DNA
vii. Criteria for Plasmid Cloning
b) Cloning Vectors Based on Bacterial Plasmids
i. Bacteriophage Vectors For E. coli
ii. Ml 3 acteriophage
iii. Cosmids
c) Vectors for Plant Cells
i. Shuttle Vectors
ii. Yeast Episomal Plasmids (YEPs)
iii. YAC Vectors
iv. Expression Vectors
d) Gene Cartridges
28. METHODS OF GENE TRANSFER
a) Bacteria and Gene Transfer
b) How Bacteria are used to Modify Plants
c) Agrobacterium Mediated Gene Transfer
i. lant-transformation Vectors
d) Gene Cloning
i. Creating Recombinant DNA
ii. DNA Ligase
iii. T DNA ligase
iv. Linkers
v. Stop Linker
vi. Adaptors
vii. Double-linkers
viii. Terminal Transferase
e) Cloning in Bacteria and Eukarydtes
i. Cloning in Bacteria
ii. Cloning in Eukaryotes
f) Amplification of DNAs by Polymerase Chain Reaction (PCR)
i. Procedure
ii. Applications of PCR
g) Gene Transfer Technology
i. Sexual Method
ii. Asexual Methods
h) Biological Delivery System
i. Disarmed Ti Vectors
ii. Vectors
i) Gene Transfer Using Particle Bombardment Technique
i. History
ii. Applications ofBiolistic
iii. Particle Gun Design
iv. Preparation of DNA-coated
Microprojectiles
j) Gene Gun
k) Modified Bombardment Devices
l) Factors Affecting Particle Bombardment-Mediated
Transformation
m) Components of the Introduced Plasmid DNA
n) Target Tissue
i. Tissue Treatment
ii. The Fate of the Introduced DNA(s)
o) Gene Transfer by Microinjection
i. Technique
ii. Applications of Microinjection
Technique
iii. The Technique
p) Silicon Carbide Fiber-Vortex Silicon Whiskers
q) Sonication
r) Electroporation
s) Poly-Cation Mediated DNA Uptake
t) Ca-DNA Co-Precipitation Method
u) Ultrasonication
v) UV Laser Microbeam
w) Genetic Transformation in Monocotyledonous Plants
x) Factors involved in Agrobacterium-mediated transfer of
genes to monocots Problems involved in gene transfer
i. Expression System
ii. Cellular Localization
iii. Proteolysis, Protein Folding and
Prosthetic Group Acquisition
iv. Precursor Availability
v. Inhibitory Environments
vi. Side Reaction of New
vii. Compounds
29. BIOTECHNOLOGY PRODUCTION OF SECONDARY PLANT METABOLITES
a) Production of Secondary
i. Metabolites in Plants
b) Stages of secondary metabolites production
i. Stress Products
ii. Genetic Engineering Products
c) Uses of Tissue Culture Techniques for Production of
Secondary
i. Plant Metabolites,
d) Applications of New Culture Methods for the Production of
Secondary Metabolites
i. Hairy Root Culture
e) Elicitation of Product Accumulation
f) Biotransformation: Production of Phrmaceutical Compounds
g) Evaluation of Antimicrobial and Antifertility Activity
h) Biological Control on Production of Secondary Metabolites
30. APPENDIX
a) Physical Quantities and Preparation of Solutions
b) Laboratory Help
c) pH and Buffers
d) Some Interesting World Wide Web Sites
31. GLOSSARY
32. SUBJECT INDEX
