M.Sc.Bio-Technology SEMESTER II-

4006 GENETICS AND MOLECULAR BIOLOGY

Unit I: Classical Vs modern genetics

History of genetics, Mendelian principles, monohybrid and dyhybrid crosses, dominance, codominance and incomplete dominance, gene interaction and epistasis, concept of gene and cistron, cis-trans complementation experiment, lethal, selfish and pseudogenes, gene concept, structure of genes.

Unit II: Structural and numerical aberrations

Ploidy level, euploidy and aneuploidy, INDELs, inversion and replacement mutations, spontaneous and induced mutation, mutagens, crossing over and linkage.

Unit III: Chromosome organization and replication

Organization of eukaryotic chromosome, gene concept, eukaryotic and prokaryotic gene architecture, Replication of DNA in prokaryotes, DNA damage and repair, Comparison of DNA replication between prokaryotes and eukaryotes.

Unit IV: Expression of gene

Gene expression in eukaryotes: Transcription, general and specific transcription factors, regulatory elements and mechanism of regulation, processing of transcripts and translation (initiation, elongation and termination) Operon concept in prokaryotes, Trp attenuation, comparison of gene expression mechanism between eukaryotes and prokaryotes.

Unit V: Gene silencing and gene expression analysis

Gene silencing approaches: cosuppression, antisense RNA techniques, ribozyme (Hammer head, hairpin ribozymes) mediated methods, dsRNA (microRNA and small interfering RNA).

TEXT AND REFERENCES:

  1. Molecular Cell Biology by Bruce Albert
  2. Molecular Biology by Lodish, Darnell and Baltimore
  3. Molecular Biology of the gene by Watson et al 4th ed.
  4. Cell and molecular biology by Gerald Karp
  5. Principles of genetics by Gardener
  6. Genetics by P.K. Gupta

4007 ENZYMOLOGY AND ENZYME TECHNOLOGY

Module I: Enzymes
Introduction and scope, nomenclature and classification of enzymes, basic mechanism of catalysis, enzyme catalysis in organic media and ionic liquids, Industrial applications.

Module II: Enzyme Kinetics
Single substrate- steady state kinetics, King-Altman’s method, Inhibitors and activators, Multi-substrate systems, Effect of pH and temperature, Allosteric enzymes. Thermodynamic explanation for transition complex formation, reaction mechanisms, Michaelis – Menten equation and its limitations, presentation of enzymatic data (direct and semilogrithmic), LB plot method to study enzyme kinetics, effect of substrate, pH and temperature on kinetics, allosteric enzyme kinetics.

Module III: Purification and immobilization of Enzymes
Enzyme isolation, disruption, fractionation, purification and concentration methods, Methods of purity estimation, purification related data presentation, advantages, Carriers, adsorption, covalent coupling, cross-linking and entrapment methods, Micro-environmental effects.

Module IV: Industrial production of enzymes
Basic concept of industrial scale and optimization, amylase, glucose oxidase, lipase, protease, production and their uses.

Module V: Challenges and future trends
Catalytic antibodies and Non-protein biomolecules as catalysts, Biocatalysts from extreme thermophillic and Hyperthermophilic Archaea and Bacteria, application of enzymes in research diagnostics and industry

TEXT AND REFERENCES:

  1. Enzyme Technology, M.F. Chaplin and C. Bucke, Cambridge University Press.
  2. Enzymes: A Practical Introduction to Structure, Mechanism and Data Analysis, R.A. Copeland, John Wiley and Sons Inc.
  3. Enzymes Biochemistry, Biotechnology, Clinical Chemistry, Trevor Palmer
  4. Enzyme Kinetics: Behaviour and Analysis of Rapid Equilibrium and Steady State Enzyme Systems, I.H. Segel, Wiley-Interscience
  5. Industrial Enzymes & their applications, H. Uhlig, John Wiley and Sons Inc

4008 IMMUNOLOGY AND STEM CELL

Unit I: Immunology- fundamental concepts and anatomy of the immune system
Components of innate and acquired immunity; Phagocytosis; Complement and Inflammatory responses; Haematopoesis; Organs and cells of the immune system- primary and secondary lymphoid organs; Lymphatic system; Lymphocyte circulation; Lymphocyte homing; Mucosal and Cutaneous associated Lymphoid tissue.(MALT&CALT); Mucosal Immunity; Antigens – immunogens, haptens; Major Histocompatibility Complex – MHC genes, MHC and immune responsiveness and disease susceptibility.

Unit II: Immune responses generated by B and T lymphocytes
Immunoglobulins-basic structure, classes & subclasses of immunoglobulins, antigenic determinants; Multigene organization of immunoglobulin genes; B-cell receptor; Immunoglobulin superfamily; Basis of self –non-self discrimination; memory; B-cell maturation, activation and differentiation; Generation of antibody diversity; T-cell maturation, activation and differentiation and T-cell receptors; Functional T Cell Subsets; Cell-mediated immune responses, ADCC; Cytokines-properties, receptors and therapeutic uses; Antigen processing and presentation- endogenous antigens, exogenous antigens, nonpeptide bacterial antigens and super-antigens; Cell-cell co-operation, Hapten-carrier system.

Unit III: Antigen-antibody interactions
Precipitation, agglutination and complement mediated immune reactions; Advanced immunological techniques – RIA, ELISA, ELISPOT assay, immunofluorescence, flow cytometry and immunoelectron microscopy; Surface plasmon resonance, Biosenor assays for assessing ligand –receptor interaction, CMI techniques- lymphoproliferation assay, Mixed lymphocyte reaction, Cell Cytotoxicity assays, Apoptosis, Microarrays, Transgenic mice, Gene knock outs.

Unit IV: Immunization
Active and passive immunization; Live, killed, attenuated, sub unit vaccines; Vaccine technology- Role and properties of adjuvants, recombinant DNA and protein based vaccines, plant-based vaccines, reverse vaccinology; Peptide vaccines, conjugate vaccines; Catalytic antibodies and generation of immunoglobulin gene libraries.

Unit V: Clinical Immunology
Immunity to Infection : Bacteria, viral, fungal and parasitic infections (with examples from each group); Hypersensitivity – Type I-IV; Autoimmunity; Types of autoimmune diseases; Mechanism and role of CD4+ T cells; MHC and TCR in autoimmunity; Treatment of autoimmune diseases; Transplantation – Immunological basis of graft rejection; Clinical transplantation and immunosuppressive therapy; Tumor immunology – Tumor antigens; Immune response to tumors and tumor evasion of the immune system, Cancer immunotherapy; Immunodeficiency-Primary immunodeficiency, Acquired or secondary immunodeficiency.

TEXT AND REFERENCES:

  1. Kuby, RA Goldsby, Thomas J. Kindt, Barbara, A. Osborne Immunology, 6th Edition, Freeman, 2002.
  2. Brostoff J, Seaddin JK, Male D, Roitt IM., Clinical Immunology, 6th Edition, Gower Medical Publishing, 2002.
  3. Janeway et al., Immunobiology, 4th Edition, Current Biology publications., 1999.
  4. Paul, Fundamental of Immunology, 4th edition, Lippencott Raven, 1999.
  5. Goding, Monoclonal antibodies, Academic Press. 1985.

4009 GENETIC ENGINEERING AND GENE THERAPY

Unit I: Basic cloning techniques
Enzymes used in cloning (Nucleases including restriction endonucleases, Polymerases, methylases, ligases, phosphatases and topoisomerases.
Vectors used in cloning and expression-Cloning vectors: Plasmids and Phage (Lambda and M13 derived) Vectors, phagemids, cosmids, artificial chromosome vectors (YAC, BAC), Animal virus derived vectors – SV40 and retroviral vectors and transposons.

Unit II: Transformation and related techniques
Competent cell preparation methods, Transformation methods for bacteria, plant and animal cells. Screening of transformants- selection markers (antibiotic resistance and genes of essential metabolism), alpha complementation for recombinant selection, reporter genes (GUS assay, luciferase), strategies for heterologous expression of genes and guiding principles.

Unit III: PCR and related techniques
Polymerase chain reaction: Thermal profile and reaction components: Optimization (touch down / hotstart and gradient PCR)
Types of PCR and their applications: Conventional PCR, AP-PCR, Anchored-PCR, Inverse-PCR, Multiplex-PCR, Reverse Transcription-PCR, DDRT-PCR and Real Time-PCR. PCR based cloning: T/A cloning, TOPO cloning and gateway cloning.

Unit IV: Construction of libraries, screening and sequencing
Construction of gene libraries: genomic DNA, subgenomic DNA, EST and cDNA libraries.
Methods of library screening: Types of probes and their construction methods, hybridization based (using radiolabelled and nonradiolabelled probes) and Immuno-screening methods.
DNA sequencing methods: Conventional and Next Generation sequencing approaches. Maxum Gilbert chemical degradation method, Sanger’s dideoxy chain termination method, Nanopore sequencing, pyrosequencing, Ion torrent method.

Unit V: Applications of genetic engineering:
Gene downregulation-using antisense RNA, dsRNA and cosuppression, CRISPR- cas 9. Site directed mutagenesis (PCR based methods) Assessment of gene functional boundary, transgenic animals (knockout mice) and plants (Flavr savr tomato), production of recombinant pharmaceuticals (insulin
and somatostatin), DNase foot printing, gene therapy (in vitro and in vivo methods). Biosafety regulation: Physical and biological containments.

TEXT AND REFERENCES:

  1. Molecular Cloning: Meniates et al
  2. Gene cloning and DNA analysis by T.A. Brown
  3. Principles of gene manipulation by Old and Primrose.
  4. Genetic engineering: Smita Rastogi and Neelam Pathak, Oxford press.

4010 LAB COURSE II

Unit I

  1. Squash preparation for chromosome staining for mitosis (onion root tip).
  2. Visualization of chromosomal translocation in Rhoeo discolour.
  3. Visualization of meiosis stages (Grasshopper testes and Vicia faba).
  4. Study of gene expression using blotting or RT-PCR.

Unit II

  1. Isolation of enzyme from plants/ bacteria.
  2. Estimation of enzyme activity and ammonium sulphate fractionation/ centrifugation based size fractionation.
  3. Determination of pH optima for an enzyme.
  4. Effect of temperature on enzymatic activity.
  5. Enzyme immobilization.

Unit III

  1. Blood cell counting.
  2. Blood group determination.
  3. Immunoprecipitation.
  4. ELISA
  5. Examine the cells comprising immune system.
  6. Trypan blue cell exclusion test for viability.

Unit IV

  1. Isolation of genomic and plasmid DNA.
  2. PCR based amplification of DNA.
  3. Cloning of PCR product in vector using T/A cloning strategy.
  4. Preparation of competent cells and its transformation with PCR product ligated in vector.
  5. Screening of recombinant transformants using blue white selection and confirmation of clone.