- Introduction to Biotechnology
- historical perspective
- biblical times
- L.Pasteur
- world wars
- penicillin
- stirred tank reactor
- mutagenesis and selection
- animal cells in culture
- monoclonal antibodies
- vaccine production
- therapeutic proteins
- plant cells in culture
- recombinant DNA technology and genetic engineering
- production of recombinant protein in E.coli (insulin)
- gene therapy
- transgenesis
- DNA vaccines
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- Recombinant DNA Technology
- clone
- cloning
- plasmids
- restricition enzymes - EcoRI, Bam HI, etc.
- sticky overhangs
- blunt ends
- annealing
- ligation - ligase
- pBR322
- origin of replication
- small
- unique cloning sites
- selectable markers - antibiotic resistance genes
- genomic library
- pUC plasmids
- antibiotic resistance gene
- lac Z gene
- pKIL plasmid
- gene expression in prokaryotes
- transcription
- gene - definition
- promoter: -10 and -35 boxes
- RNA polymerase
- transcription initiation complex
- localized 'melting'
- primary transcript
- ribosome-binding site-- Shine-Dalgarno sequence
- gene expression in eukaryotes
- RNA polymerases
- promoter
- promoter proximal sequences
- enhancers
- pre-messenger RNA
- processing or maturation
- cappping
- polyadenylation [poly A-tailing]
- splicing
- introns and exons
- posttranslational modifications
- complementary DNA (cDNA)
- messenger RNA
- primer
- reverse transcriptase
- cDNA library
- optimizing expression of eukaryotic genes in prokaryotes
- screening
- genetic complementarity
- nucleic acid hybridization
- immunological detection
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- Recombinant DNA technology
- Applications
- labeling DNA probes
- DNA polymerase E. coli
- activities
- Klenow fragment
- nick-translation labeling
- random primer labeling
- non-isotopic (chemiluminescence) labeling
- Southern analysis
- DNA isolation
- restriction enzyme digest
- agarose gel electrophoresis
- denaturation
- blotting
- blocking
- hybridization (radiolabelled or chemiluminescent probe)
- washing
- detection
- screening for genetic disorders
- oligonucleotide probes
- alpha-antitrypsin deficiency
- restriction fragment length polymorphisms RFLP
- alpha thalassemia
- sickle cell anemia
- DNA fingerprinting
- minisatellite DNA
- oligonucleotide 'core' probes
- paternity disputes
- homicide
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- Protein Engineering
- enzymes and industry
- site-directed mutagenesis
- M13 bacteriophage
- structure
- replication cycle
- plus strand
- replicative form (RF)
- genetic map
- cloning vector
- polycloning site
- oligonucleotide directed mutagenesis
- thionucleotide selection
- applications
- increased thermal stability - lysozyme
- increased specific activity - interferon
- resistance to oxidative damage - alpha-antitrypsin and subtilisin
- fusion proteins with new pharmacological properties
- tumor necrosis factor/interferon
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- Large Scale Cultivation
of Microbes
- growth kinetics
- industrial fermentation
- delivery of O2 - solubility properties
- stirred tank reactor
- vessel
- sparger
- agitator
- cooling jackets/coils
- pH control
- antifoam
- fermenter train
- operation
- airlifter fermenter
- deep-shaft reactor
- downstream processing
- harvesting - centrifgugation, ultrafiltration
- cell disruption - high pressure homogenizer, high-speed mill
- protein purification
- precipitation
- chromatography
- size exclusion/gel filtration
- ion exchange
- affinity
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- Commercial
Exploitation of Microorganisms
- production of biomass
- Penicillium roquefortii
- lactic acid bacteria
- Rhizobium
- Pseudomonas syringae
- Bacillus thuringensis
- single cell protein
- Methylophilus methylotrophus
- Pichia
- bacterial vaccines
- suspension of killed bacteria
- live, attenuated bacteria
- subunit vaccines
- production of low molecular weight compounds
- secondary metabolites
- bioconversions or biotransformations
- indigo
- vitamin C
- cortisone
- semisynthetic opiates
- production of high molecular weight compounds
- proteins
- bulk enzymes
- recombinant proteins
- polysaccharides
- xantham gum
- gellan
- emulsan
- pullulan
- dextrans
- good manufacturing practice [GMP]
- quality control
- quality assurance
- processes dependent on general microbial metabolism
- bioremediation
- molecular breeding - oil and 2,4,5-T degrading bacteria
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- Cell and Enzyme Immobilization
- rationale
- immobilization methods
- physical binding
- crosslinking
- entrapment
- choice of method
- stability
- activity
- regenerability
- reactor configuration
- cost of catalyst
- examples
- L-amino acid acylase
- high fructose syrup
- 6-aminopenicillanic acid
- urocanic acid
- malic acid
- aspartic acid
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- Mammalian Cell Culture
- methodology
- tissue explant
- cell suspension
- primary culture
- fibroblasts
- epithelial cells
- anchorage dependent
- cell strain
- cell line
- neoplastic transformation
- media
- balanced salt solutions - Earle and Hanks
- supplements
- modes of cell growth
- anchorage-dependent
- suspension culture
- colonies on semisolid medium - hybridomas
- large scale cultivation
- roller bottles
- microcarrier beads
- hollow fiber apparatus
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- Applications of
Animal Cell Culture
- toxicity testing
- production of viral vaccines
- methodology
- live, avirulent
- killed (inactivated)
- subunit vaccines
- peptide vaccines
- production of therapeutic proteins
- overproduction by mammalian cell lines
- cloning in mammaliam cells
- transfection [direct gene transfer techniques]
- Ca-phosphate/DNA coprecipitates
- lipofection
- electroporation
- microinjection
- viral vectors
- advantages and limitations
- vaccinia virus
- retrovirus
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- Transgenesis and Gene
Therapy
- definitions
- germ cells vs. somatic cells
- examples of transgenesis
- basic protocol
- microinjection of eggs
- reimplantation
- transgenic 'supermouse'
- transgenic salmon
- transgenic farm animals
- lessons from transgenic pigs
- biopharming - mammary glands as bioreactors
- examples of gene therapy
- adenine deaminase
- hemophilia B dog
- HSV thymidine kinase gene as suicide gene for cancer treatment
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- Monoclonal Antibodies
- basic immunology
- definitions - innate vs specific immunity
- B-lymphocytes and T-lymphocytes - origins
- antigen/ antigenic determinant
- antibody
- cell mediatd immunity
- antibody mediated immunity
- virgin or resting B-cells
- memory cells
- plasma cells
- origin of antibody specificity
- instructive vs. clonal selection theory
- methodology
- lymphocytes
- multiple myeloma cells
- cell fusion - PEG
- hybridoma
- selection
- large scale production of Mabs
- roller bottles
- spinner bottles
- ascites
- microencapsulation
- applications of Mabs
- diagnostic reagents
- protein purification by affinity chromatography
- bone marrow transplantation
- donor/recipient typing
- graft-vs-host disease
- radioimmunotherapy
- diagnostic imaging
- bispecific antibodies
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- Genetic Engineering
in Plants
- major goals
- Agrobacterium tumefaciens-mediated gene transfer
- dicot plants (vs monocots)
- crown galls
- Ti-plasmid
- T-DNA
- oncogenes - auxin and cytokinin synthesis genes
- opine synthesis genes
- opine catabolism genes
- virulence genes
- callus
- 'disarming' the Ti-plasmid
- regeneration of transgenic plants
- totipotency
- leaf disc method
- some achievements
- resistance to viral infections - Tobacco Mosaic V.
- resistance to insect attacks - B.t. toxin
- reistance to herbicides - bromxynil
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- Antisense Oligonucleotide Technology
- contain spread of viral infections
- treatment of cancer
- downregulating the synthesis of allergens in plants
- challenges
- targetting
- uptake
- prevention of nuclease attack
- phosphorothioate
- phosphoramidate
- peptide nucleic acid
- specificity
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Human Genome Project
- objectives
- costs
- phases
- benefits and controversies
- global gene expression
- microarray analysis
- applications
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