M.Sc.Microbiology Semester IV-

TH – XIII RECOMBINANT DNA TECHNOLOGY

Unit – 1 Techniques and enzymes in genetic recombination
Core techniques and essential enzymes used in recombination: restriction endonucleases, type I, II, III, recognition sequences, properties, nomenclature, classification of type II endonucleases, their activity. DNA ligase: Properties and specificity, S1 nuclease, BAL 31 nuclease, DNA polymerase, polynucleotide kinase, phosphatase, reverse transcriptase its activity and mode of action. Chemical synthesis of DNA. Restriction digestion, ligation and transformation.

Unit – 2 Plasmids
Properties, incompatibility, isolation and purification techniques, plasmid vectors and their properties, PBR 322 – its construction and derivatives, single stranded plasmids, promoter probe vectors, runaway plasmid vectors.
Bacteriophage lambda () as a vector: Essential features, organization of  genome, generalstructure, rationale for vector construction, improved  vectors,  gt series,  EMBL vectors, invitro packaging, cosmids, phasmids, filamentous phage vectors,  zap,  blue print vectors.

Unit- 3 Specialized cloning strategies
Expression vectors, promoter probe vectors, vectors for library construction, genomic DNA libraries, chromosome walking and jumping, cDNA libraries, short gun cloning, directed cloning, phage display. Recombinant DNA technology with reference to cloning and production of interferon and insulin. Miscellaneous applications of Genetically engineered micro organisms (GEMS) / genetically modified organisms (GMO’s).

Unit – 4 PCR methods and Applications
PCR methods and Applications DNA sequencing methods, Dideoxy and Chemical method. Sequence assembly. Automated sequencing.

Unit – 5 Molecular mapping of genome
Genetic and physical maps, physical mapping and map –based cloning, choice of mapping population, simple sequence repeat loci, southern and fluorescence in situ hybridization for genome analysis, Chromosome microdissection and microcloning, molecular markers in genome analysis:
RFLP, RAPD and AFLP analysis, molecular markers linked to disease resistance genes, Application of RFLP in forensic, disease prognosis, genetic counseling, pedigree, varietal etc. animal trafficking and poaching: Germplasm maintenance, taxonomy and Biodiversity.

References-

  1. Principles of Gene Manipulations 1994 by Old and Primrose Blackwell Scientific Publications.
  2. DNA Cloning: A Practical Approach by D.M. Glower and B.D. Hames, IRL Press, Oxford. 1995.
  3. Molecular Biotechnology 2nd Edition by S.B. Primrose. Blackwell Scientific Publishers, Oxford. 1994.
  4. Genetic Engineering and Introduction to Gene Analysis and Exploitation in Eukaryotes by S.M. Kingsman and A.J. Kingsman, Blackwell Scientific Publications, Oxford 1998.
  5. PCR Technology – Principles and Applications for DNA Amplification by Henry A. Erlich (Ed.) Stockton Press. 1989.
  6. Biotechnology: A Guide to Genetic Engineering by Peters.
  7. Genetic Engineering – 2000 by Nicholl.
  8. Recombinant DNA and Biotechnology: Guide for Teachers. 2nd Edition by Helen Kreuz. 2001.ASM Publications.
  9. Molecular Biotechnology: Principles and Applications of Recombinant DNA. 2 nd Edition. 1998 by Bernard R. Glick and Jack J. Pastemak, ASM Publications.
  10. From genes to clones by Winnaker.
  11. Manipulations and expression of recombinant DNA by Robertson.
  12. Gene targeting – A practical approach by Joyner.

TH-XIV FERMENTATION TECHNOLOGY

Unit – 1 Microbial Fermentations
Metabolic pathways and metabolic control mechanisms, industrial production of citric acid, lactic acid, enzymes (alpha-amylase, lipase, xylase, pectinases, proteases), acetone- butanol, lysine and glutamic acid.

Unit – 2 Microbial production of therapeutic compounds
Microbial production of therapeutic compounds ( lactam, aminoglycosides, Ansamycins (Rifamycin), peptide antibiotics Quinolinones), biotransformation of steroids, vitamin B12 and riboflavin fermentation.

Unit – 3 Modern trends in microbial production
Modern trends in microbial production of bioplastics (PHB, PHA), bioinsectices (thuricide), biopolymer (dextran, alginate, xanthan, pullulan), Biofertilizers (nitrogen fixer Azotobacter, Phosphate solubilizing microorganisms), Single Cell Protein and production of biological weapons with reference to anthrax.

Unit – 4 Biofuels
Useful features of bio-fuels. The substrate digester and the microorganisms in the process ofbiogas production (biomethanation). Production of bioethanol from sugar, molasses, starch and cellulosic materials. Ethanol recovery. Microbial production of hydrogen gas, biodiesel from hydrocarbons.

Unit – 5 Immobilization techniques , IPR and Patents
Some industrial techniques for whole cell and enzyme immobilization. Application and advantages of cell and enzyme immobilization in pharmaceutical, food and fine chemical industries. Intellectual Property Rights (IPR), Patents, Trademarks, Copyrights, Secrets, Patenting of biological materials, international co operation, obligations with patent applications, implicationof patenting, current issues, hybridoma technology etc. Patenting of higher plants and animals, transgenic organisms and isolated genes, patenting of genes and DNA sequences, plant breeders right and farmers rights.

References-

  1. Biotechnological Innovations in Chemical Synthesis. BIOTOL. Publishers / Butterworth Heinemann.
  2. Industrial Microbiology by G. Reed (Ed), CBS Publishers (AVI Publishing Co.)
  3. Biology of Industrial Microorganisms by A.L. Demain.
  4. Genetics and Biotechnology of Industrial Microorgansims by C.l. Hershnergey, S.W. Queener and Q. Hegeman. Publisher. ASM. Ewesis ET. Al. 1998. Bioremediation Principles. Mac Graw Hill.
  5. Annual Reports in Fermentation Processes by D. Pearlman, Academic Press.
  6. Fundamentals of Biochemical Engineering by Bailey and Ollis.
  7. Annual Review of Microbiology by Charles E. Cliffton (Volumes)
  8. Biotechnology, A textbook of industrial Microbiology by Creuger and Creuger, Sinaeur associates.
  9. Manual of industrial Microbiology and Biotechnology 2nd edition by Davis J.E. and Demain A.L. ASM publications.

TH-XV BIOINFORMATICS, MICROBIAL GENOMICS AND PROTEOMICS.

Unit – 1 Bioinformatics and its applications
Databases, types, pairwise and multiple alignments. Structure-function relationship. Sequence assembling using computers. Computer applications in molecular biology, Protein domains and human genome analysis program (BLAST, FASTA, GCC etc.) Search and retrieval of biological information and databases sequence, databank. (PDB and gene bank), accessing information (Network expasy, EMB Net, ICGEB Net).

Unit – 2 Whole genome analysis
Preparation of ordered cosmid libraries, bacterial artificial chromosomal libraries, shotgun libraries and sequencing, conventional sequencing (Sanger, Maxam and Gilbert Methods), automated sequencing.

UNIT – III Sequence analysis
Computational methods, homology algorithms (BLAST) for proteins and nucleic acids, open reading frames, annotations of genes, conserved protein motifs related structure / function (PROSITE, PFAM, Profile Scan). DNA analyses for repeats (Direct and inverted), palindromes, folding programmes. Use of Internet, public domain databases for nucleic acid and protein sequences (EMBL, GeneBank), database for protein structure (PDB).

UNIT – IV DNA Microarray
Printing or oligonucleotides and PCR products on glass slides, nitrocellulose paper. Whole genome analysis for Global patterns of gene expression using fluorescent-labelled cDNA or end labelled RNA probes. Analyses of single nucleotide polymorphism using DNA chips.

UNIT – V Proteome analysis
Two dimensional separation of total cellular proteins, isolation and sequence analysis of individual protein spots by Mass Spectroscopy. Protein microarrary advantages and disadvantages of DNA and protein microarrays.

References-

  1. Bioinformatics. 1998 by Baxevanis
  2. Bioinformatics 2000 by Higgins and Taylor OUP.
  3. Nucleic acid Research 2001. Jan. Genome database issue.
  4. The Internet and the new Biology: Tools for Genomics and Molecular Research by Peruski, Jr. and Peruske (ASM) 1997.
  5. Functional Genomics. A Practical Approach Edited by Stephen P Hunt and Rick Liveey (OUP) 2000.
  6. DNA microarrays: A practical approach edited by Mark Schena (OUP)
  7. Bioinformatics – A Practical Guide to the Analysis of Genes and Proteins. 2nd Edition by Baxevanis.
  8. Bioinformatics: Sequence, structure and Data Bank: A Practical Approach by Higgis.
  9. Bioinformatics – from Genomes to drug. 2 volumes by Lenganer.
  10. Bioinformatic Methods and Protocols – Misener.
  11. Bioinformatics: Sequence and Genome analysis.
  12. Introduction to Bioinformatics by Altwood.
  13. Proteome Research: New Frontiers in Functional Genomics: Principles and Practices.
  14. Genomics: The Science and Technology behind the human project.
  15. Protein Biotechnology. Edited by Felix Franks. Humana Press, Totowa, New Jarsey.
  16. Protein Engineering: Principles and Practice by Cleland.
  17. Computer analysis of sequence data by Colte.
  18. Web sites for Proteomics and Genomics
    www.geneprot.com, www.hybrigenis.com, www.mdsproteomics.com, www.stromix.com, www.syrrx.com.

TH –XVI PHARMACEUTICAL MICROBIOLOGY

Unit – 1 Antibiotics and synthetic antimicrobial agents
Antibiotics and synthetic antimicrobial agents (Aminoglycosides,  lactams, tetracyclines, ansamycins, macrolid antibiotics) Antifungal antibiotics, antitumor substances. Peptide antibiotics, Chloramphenicol, Sulphonamides and Quinolinone antimicrobial agents. Chemical disinfectants, antiseptics and preservatives.

Unit – 2 Mechanism of action of antibiotics
Mechanism of action of antibiotics (inhibitors of cell wall synthesis, nucleic acid and protein synthesis).
Molecular principles of drug targeting.
Drug delivery system in gene therapy
Bacterial resistance to antibiotics.
Mode of action of bacterial killing by quinolinones.
Bacterial resistance to quionolinones.
Mode of action of non – antibiotic antimicrobial agents.
Penetrating defenses – How the antimicrobial agents reach the targets (cellular permeability barrier, cellular transport system and drug diffusion).

Unit – 3 Microbial production and Spoilage of pharmaceutical Products
Microbial contamination and spoilage of pharmaceutical products (sterile injectibles, non injectibles, ophthalmic preparations and implants) and their sterilization. Manufacturing procedures and in process control of pharmaceuticals. Other pharmaceuticals produced by microbial fermentations (streptokinase, streptodornase). New vaccine technology, DNA vaccines, synthetic peptide vaccines, multivalent subunit vaccines. Vaccine clinical trials.

Unit – 4 Regulatory practices, biosensors and applications in Pharmaceuticals
Financing R&D capital and market outlook. IP, BP, USP.
Government regulatory practices and policies, FDA perspective.
Reimbursement of drugs and biologicals, legislative perspective.
Rational drug design.
Immobilization procedures for pharmaceutical applications (liposomes).
Macromolecular, cellular and synthetic drug carriers.
Biosensors in pharmaceuticals.
Application of microbial enzymes in pharmaceuticals.

Unit – 5 Quality Assurance and Validation
Good Manufacturing Practices (GMP) and Good Laboratory Practices (GLP) in pharmaceutical industry.
Regulatory aspects of quality control.
Quality assurance and quality management in pharmaceuticals ISO, WHO and US certification.
Sterilization control and sterility testing (heat sterilization, D value, z value, survival curve, Radiation, gaseous and filter sterilization)
Chemical and biological indicators.
Design and layout of sterile product manufacturing unit. (Designing of Microbiology laboratory)
Safety in microbiology laboratory.

References-

  1. Pharmaceutical Microbiology – Edt. by W.B.Hugo & A.D.Russell Sixth edition. Blackwell scientific Publications.
  2. Analytical Microbiology –Edt by Frederick Kavanagh Volume I & II. Academic Press New York.
  3. Quinolinone antimicrobial agents – Edt. by David C. Hooper, John S.Wolfson .ASM Washington DC.
  4. Quality control in the Pharmaceutical Industry – Edt. by Murray S.Cooper Vol.2. Academic Press New York.
  5. Biotechnology – Edt. by H.J.Rehm & G.Reed, Vol 4. VCH Publications, Federal Republic of Germany.
  6. Pharmaceutical Biotechnology by S.P.Vyas & V.K.Dixit. CBS Publishers & Distributors, New Delhi.
  7. Good Manufacturing Practices for Pharmaceuticals Second Edition, by Sydney H.Willig, Murray M.Tuckerman, William S.Hitchings IV. Mercel Dekker NC New York.
  8. Advances in Applied Biotechnology Series Vol 10, Biopharmaceuticals in transition. Industrial Biotechnology Association by Paine Webber. Gulf Publishing Company Houston.
  9. Drug Carriers in biology & Medicine Edt. by Gregory Gregoriadis. Academic Press New York.
  10. Quality Assurance in Microbiology by Rajesh Bhatia, Rattan lal
  11. Ihhpunjani. CBS Publishers & Distributors, New Delhi.