Course Objectives:
a. To learn about the basic components of molecular cloning
b. Understand molecular techniques related to genetic engineering
c. To learn nucleic acid and protein blotting techniques
d. To comprehend latest molecular biology techniques and their applications
2. Course Outcomes: (C.O):
a. Understanding the fundamentals of cloning processes and components involved
b. Comprehension of various molecular biological techniques and their applications
c. Acquaintance with recombinant DNA and DNA sequencing techniques
d. Knowledge regarding the use of rDNA techniques in modern biology, medicine and agriculture
Course Plan
Unit I Fundamentals of rDNA Technology
1. Restriction Enzymes A. Host controlled restriction modification B. Exonucleases and Endonucleases C. Classification & applications of Restriction Enzymes
2. DNA modifying enzymes A. Methylases &Polymerases B. Ligases & Kinases C. Phosphatases & Nucleases
3. Vectors in RDNA Technology A. Salient features of vectors B. Types of vectors – plasmids, phages, shuttle vectors, phagemids C. Significance of vectors in rDNA Technology
4. Recombinant Vectors in rDNA Technology A. Cloning vectors – pBR322, pUC 19, pTZ vector B. Expression vectors – pET & pGEX vector C. Vectors for Library preparation – phage vectors (λ phage), Cosmids, BAC, YAC
5. Hosts in RDNA Technology A. Bacteria – E. coli, Yeast – Saccharomyces cerevisiae B. Plant and Animal Cells C. Insect & Mammalian cell lines
Unit II Cloning and selection of recombinant clones
1. Introduction to Cloning A. Traditional cloning – advantages & disadvantages B. PCR based cloning – its significance C. Recombination based cloning – Advantages
2. Construction of Genomic DNA library A. Strategies for construction of Genomic DNA library B. Colony hybridization & library screening C. Chromosome Walking & Chromosome Jumping; Positional cloning of genes
3. Construction of cDNA library A. Strategies for construction of cDNA library B. Construction of subtractive normalized cDNA libraries C. PCR- principle, types (multiplex, nested and touch-down PCR) and applications
4. Selection and characterization of recombinant clones I A. Genetic Selection - insertional inactivation and alpha complementation B. Labeling of nucleic acids C. Immunological probes
5. Selection and characterization of recombinant clones II A. Selection of recombinant clones-hybridization techniques (Southern, Northern, Western, South-Western & Zoo blot) B. Hybrid arrest & Hybrid release translation C. Characterization using DNA sequencing methods- Maxam-Gilbert and Sanger’s method
Unit III Advances & Applications of RDNA Technology
1. Advances in RDNA Technology I A. Site directed mutagenesis B. Genetic engineering technologies C. RNA interference
2. Advances in RDNA Technology II A. Knock in and Knock out technology B. Genome Editing technologies C. Next Generation Sequencing
3. Applications of RDNA Technology I A. Applications in Agriculture – Bt Cotton, Herbicide resistant crops B. Applications in Horticulture C. Applications in Animal Husbandry
4. Applications of RDNA Technology II A. Production of Therapeutics – human insulin, monoclonal antibodies, interferons, human growth hormone, blood clotting factor VIII B. Vaccine development – Recombinant vaccines (Hepatitis B vaccine), cDNA vaccines C. Applications in Molecular diagnostics – Real Time PCR tests for HIV, COVID
5. Applications of RDNA Technology III A. DNA Fingerprinting B. Strain improvement and production of antibiotics C. Applications in Bioremediation
PRACTICALS BT 351 P: RECOMBINANT DNA TECHNOLOGY
Unit I
1. Isolation of a gene using PCR and agarose gel electrophoresis analysis
2. Isolation of plasmid DNA (vector) and agarose gel electrophoresis analysis
3. Restriction digestion of insert (Gene amplicon) and vector (plasmid DNA)
4. Gel elution of digested insert or vector DNA
5. Ligation of insert (Gene amplicon) and vector (plasmid DNA)
6. Preparation of competent cells of E. coli for transformation
7. Transformation of E. coli and selection of transformed colonies
Unit II
1. Selection of recombinant clones using colony PCR
2. Confirmation of recombinant clones using double digestion of recombinant clones
3. Confirmation of recombinant clones using DNA sequencing
4. Cloning & expression of a gene using pET expression vector system
5. Cloning & expression of a gene using GATEWAY cloning system
6. Whole plasmid mutagenesis (site directed mutagenesis) for point mutation
7. Restriction mapping and problems
8. Designing of guide RNA target using online tools for CRISPR CAS 9 mediated genome editing
REFERENCE BOOKS
1. Principles of Gene Manipulation and Genomics- Sandy B. Primrose, Richard Twyman 7th Edition; Blackwell Publishing
2. Gene Cloning and DNA Analysis: An Introduction- T. A. Brown - John Wiley & Sons
3. An Introduction to Genetic Engineering- Desmond S.T. Nicholl – Cambridge University Press
4. Molecular Biotechnology: Principles and Applications of Recombinant DNA -Bernard R. Glick, Jack J. Pasternak, Cheryl L. Patten- ASM Press
5. Molecular Cloning: A Laboratory Manual (Cold Spring Harbor)- M. R. Green, J.Sambrook
Course Objectives:
a) To introduce Bioinformatics and accomplish solid understanding of scope and its applications.
b) To explain the underlying basic principles of sequence analysis, and apply the same for analyzing nucleic acid and protein sequences.
c) To provide an introduction to advanced areas such as sequencing, gene identification, genome wide analysis and drug discovery.
d) To impart an in depth understanding of proteomics.
Course Outcomes:
a) Enable to explore bioinformatics web portals, databases and tools.
b) Gains familiarity in sequence comparisons with algorithms and matrices
c) Able to perform various in silico analysis for gene structure and function prediction, target identification for drug designing
d) Gain insights to interpret the output from genomic, proteomic tools to use in agriculture and medical research
Unit-I
Introduction to Bioinformatics 1.
1 Introduction to Bioinformatics: A) A historical perspective, B) aim, scope, sub-fields, C) an overview of bioinformatics applications.
1.2 Bioinformatics databases: A) What are databases, why databases, classification of databases. B) Primary and secondary databases: Nucleotide databases-Genbank, ENA, DDBJ, Protein databases- Uniprot, PDB, CATH, SCOP, metabolic pathway database-KEGG, genome variation database-dbSNP C) Specialized databases -RAP-DB, Explore and download protein structures from PDB and MMDB databases MGD
1.3 File formats: A) GenBank, FASTA, SAM, BAM, FASTQ B) ENA & NBRF C) PDB File
1.4 Bioinformatics web portals: A) NCBI B) EBI C) Expasy 1.5 Search engines and Software packages: A) Database search engines (Entrez and SRS) B) Bioinformatics online tools C) Bioinformatics commercial Software packages
Unit-II Sequence alignment and algorithms
2.1 Basics of sequence alignment: A) Match, mismatch, gap, gap penalties (linear & affine gap penalties) B) Sequence identity & similarity C) Sequence relationships (homologs, orthologs, paralogs & xenologs)
2.2 Pairwise alignment: A) Dot-matrix comparison of sequences B) Dynamic programming algorithms: Global- Needleman and Wunch algorithm C) Local- Smith and Waterman algorithm.
2.3 Scoring matrices: A) PAM B) BLOSUM C) DNA vs protein sequence alignment (permissible replacements, similarity score).
2.4 Heuristic algorithms for database searching: A) FASTA algorithm B) BLAST algorithm C) PSI BLAST
2.5 Multiple Sequence Alignment (MSA): A) Significance of MSA B) Progressive and iterative based algorithms for multiple sequence alignment C) Phylogenetic analysis- Distance matrix-based tree construction, UPGMA based tree construction
Unit-III Applications of Bioinformatics:
3.1 Bioinformatics for genome sequencing: A) First, next generation Sequencing B) Third generation Sequencing C) RNA sequencing
3.2 Genome annotation: A) Genome browsers, finding repeats B) Gene prediction in prokaryotes and eukaryotes C) Promoters and regulatory motif prediction
3.3 Protein profiling: A) 2D gel electrophoresis B) Peptide mass fingerprinting C) Protein identification
3.4 Structural bioinformatics: A) Secondary structure prediction B) Tertiary structure prediction (homology modelling) C) 3D structure evaluation
3.5 Medical & Agricultural application of bioinformatics: A) Identification of disease-causing genes B) Overview of drug discovery C) Pharmacogenomics
BIOINFORMATICS PRACTICALS:
UNIT-1 Data Retrieval& Sequence analysis:
1. Understanding DNA/protein coding systems: A) Download and install Bioedit.
2 Bioinformatics web portals A) NCBI B) EBI C) Expasy
3 Databases A) Explore and download nucleotide sequences from Genbank and ENA databases B) Explore gene databases (entrez gene, gene cards) C) Explore and download protein sequence from Uniprot database
4 File formats A) GenBank, ENA, FASTA, PDB, FastQ, BAM) B) Sequence format conversion using Readseq tool
5 Pairwise alignment of DNA and protein sequences A) Dot matrix comparison of sequences using graphs and dot matcher tool B) Global alignment- Emboss Needle C) Local alignment- Emboss Water
6 Database search tools A) FASTA B) BLAST
7. Multiple sequence alignment of DNA and protein sequences A) Clustal Omega UNIT-2 Genomics, Proteomics& Structure prediction
8. Genome browsers and databases A) UCSC genome browser B) Genome specific databases: RAP-DB, MGD C) Genome variation database - dbSNP
9. Prediction of genes in prokaryotic and eukaryotic genomes A) ORF Finder B) Genscan C) Glimmer
10. Prediction of SSRs in DNA sequence (SSRit)
11. Translation of a nucleotide (DNA/RNA) sequence to a protein sequence using Translate tool
12. Structure databases A) Explore and download protein structures from PDB B) Explore and download protein structures from MMDB
13. Compute physical and chemical parameters of protein using A) Protparam tool B) Protein identification C) Prosite
14. Prediction of secondary structures of proteins A) Chou fasman method B) GOR IV method C) Psipred
15. Tertiary structure prediction by homology model building
REFERENCE BOOKS
1. Introduction to bioinformatics by Aurther M lesk
2. Developing bioinformatics computer skills by Cynthia Gibas, Per Jambeck
3. Chemoinformatics: a textbook by Johann Gasteiger
4. Bioinformatics second edition by David M mount
5. Essential bioinformatics by Jin Xiong
6. Bioinformatics computing by Bryan Bergeron
7. Bioinformatics: concepts, skills & applications by R.S. Rastogi 8. Bioinformatics: methods and applications genomics, proteomics and drug discoveryby S.C. Rastogi, ParagRastogi, NamitaMendiratta
PAPER-III A(ELECTIVE) BT 303 T (A) - ADVANCES IN PLANT BIOTECHNOLOGY
1.Course Objectives: (C.Obj.)
a. To acquaint the principles & techniques of plant tissue culture and their applications
b. To introduce in vitro plant cell & tissue cultures and associated techniques for enhanced production of secondary metabolites
c. To acquaint the principles & techniques of plant transgenic technology with respect to genetic transformation, detection and characterization of transformants, gene expression and gene silencing; gene editing techniques
d. To illustrate the applications of plant transgenic technology
2. Course Outcomes: (C.O)
a. Understanding the aspects of in vitro regeneration and applications related to crop improvement and plant germplasm conservation
b. Critically understanding the advantages of in vitro plant cell & tissue cultures and associated techniques for enhanced production of secondary metabolites
c. Understanding the principles and techniques of plant transgenic technology, gene silencing and gene editing techniques
d. Critically understanding the applications & limitations of plant transgenic technology in developing crops that are biotic & abiotic stress tolerant, with enhanced nutritional quality and improved post-harvest qualities and producing therapeutic compounds etc.
Course Plan Unit I Plant Tissue Culture Techniques & Applications
1. Introduction to Plant Tissue Culture A. Plant Cell Totipotency, Dedifferentiation, Redifferentiation, Plant Morphogenesis B. Media components – Macroelements & Microelements, Media additives, Sterilization of media, Types of media – White’s media, B5 media, MS media; Growth regulators – Auxins, Cytokinins, Gibberlines, Abscisic acid, Ethylene C. Initiation of Callus cultures & Cell suspension cultures and Plant regeneration
2. Micropropagation and Somaclonal Variation A. Meristem culture and its applications (virus free plants) B. Micropropagation – process, methods, advantages and applications C. Somaclonal Variation - causes, molecular basis and applications
3. Haploids, Dihaploids & Embryo culture A. Production of haploids – Anther culture, Pollen culture B. Dihaploids production & their applications C. Embryo culture and Embryo rescue
4. Protoplast culture & Somatic Hybridization A. Isolation and culture of protoplasts; Fusion of protoplasts (Homokaryones & Heterokaryones) B. Somatic hybridization, selection & characterization of hybrids, their applications C. Production of Cybrids and their applications
5. Cryopreservation for conservation of plant germplasm A. Conservation of plant germplasm & its significance B. In vitro techniques for conservation of plant germplasm C. Cryopreservation – different methods, advantages & applications
Unit II In vitro plant cultures for production of secondary metabolites
1. In vitro plant cultures as source of secondary metabolites A. Plant secondary metabolites – classification & uses B. Advantages of in vitro plant cell & tissue cultures as source of secondary metabolites C. Biotransformation and production of novel compounds
2. Factors affecting the in vitro production of plant secondary metabolites A. Chemical Factors – media, nutrients, Growth regulators, Precursor feeding B. Physical Factors – Light, pH, Temperature, gaseous environment C. Biological Factors – Stages of cell cultures, Degree of differentiation
3. Strategies for enhanced production of secondary metabolites A. Permeabilization for enhanced production of secondary metabolites B. Elicitation (Abiotic & Biotic Elicitors) for enhanced production of secondary metabolites C. Immobilization for enhanced production of secondary metabolites
4. Organ cultures for enhanced production of secondary metabolites A. Adventitious shoots & roots B. Hairy root cultures C. Leafy & Shooty dermatomes
5. Pathway engineering for enhanced production of secondary metabolites A. Strategies involved in pathway engineering B. Applications and advantages C. Biosafety guidelines for pathway engineering
Unit III Genetic modification techniques and applications
1. Gene transfer techniques A. Agrobacterium mediated gene transfer technique – Ti plasmid, molecular basis of Agrobacterium infection; Binary & Cointegrate vectors B. Direct gene transfer techniques – Particle Bombardment, Electroporation, Microinjection C. Virus mediated gene transfer – VIGS
2. Selection & Characterization of genetically modified plants A. Screening of transformants using reporter genes – GUS, GFP B. Selection of transformants using selection marker genes – antibiotic (npt II, hpt II) or herbicide (bar, ppt) resistance marker genes C. Characterization of transformants using PCR & Southern blotting techniques
3. Advanced genetic modification techniques A. Chloroplast transformation – advantages & applications B. RNAi Technology – role in crop improvement C. Genome Editing Technique – CRISPR CAS 9
4. Applications of genetically modified plants I A. Abiotic stress tolerance – Light, Heat & Cold, Drought, Salinity and Heavy metal stress tolerance B. Biotic stress – Bacterial, Fungal & Viral resistance C. Insect/Pest and Herbicide resistance
5. Applications of genetically modified plants II A. Production of crops with improved growth & productivity B. Production of biopharmaceuticals – therapeutics (Insulin, Interferons, Growth hormones) & Vaccines (Edible vaccines) C. Production of crops with improved nutrition (amino acids, oils, vitamins & micronutrients)
PRACTICALS BT 353 P (A): ADVANCES IN PLANT BIOTECHNOLOGY
1. Surface sterilization of explants, inoculation and induction of callus from the explants
2. Induction of somatic embryogenesis & preparation of synthetic seeds
3. Micropropagation of an elite agricultural/horticultural plant species
4. Induction of hairy root cultures using Agrobacterium rhizogenes
5. Genetic transformation of desired plant tissue using Agrobacterium tumefaciens
6. Genetic transformation of desired plant tissue using Particle bombardment
7. Confirmation of putative genetically transformed plants using PCR/Southern blotting
8. Evaluation of an antioxidative enzyme activity in genetically transformed plants for drought/salinity stress tolerance
REFERENCE BOOKS
1. Plant tissue culture and its biotechnological applications by W. Barz, E. Reinhard, M.H. Zenk
2. Plant tissue culture by Akio Fujiwara
3. Frontiers of plant tissue culture by Trevor A. Thorpe
4. In vitro haploid production in higher plants by S. Mohan Jain, S.K. Sopory, R.E. Veilleux
5. Plant tissue culture: theory and practice by S.S. Bhojwani and A. Razdan
6. Plant cell, tissue and organ culture, applied and fundamental aspects by Y.P.S. Bajaj and A. Reinhard
1. Course Objectives (C.Obj):
a. To understand the nature and scope of Food Biotechnology.
b. To gain knowledge and understanding of food fermentation processes.
c. To comprehend role of biotechnology in the production of microbial, plant and animal-based food products and their applications.
2. Course Outcomes (C.O):
a. to acquire skills in manipulation of microorganisms, plants and animals for food production.
b. to acquaint with the methods and operations used in Food Biotechnology.
c. to apply Food Biotechnology skills for the production of industrial food produc
Course Plan/Schedule
UNIT 1: Fundamentals of Food Biotechnology
1.1 A) Role of Biotechnology in food industry; B) Impact of Biotechnology on food production; C) Food quality – quality control and importance of quality assurance
1.2 A) Microorganisms associated with food; B) Factors influencing microbial activity; C) Importance of microorganisms in food industry.
1.3 A) Crosslinking of proteins – Maillard reaction, role of cross-linking enzymes in meat; B) Protein structure, rheology and texturization; C) Dietary and Nutrient Recommendations.
1.4 A) Enzymes in food and feed; B) Physical and chemical factors affecting fermentation in food processing; C) Tools to predict physico-chemical properties of industrial food and cultivation media.
1.5 A) Carbon footprint of food; B) Applications of nanotechnology – biosensors to detect nutrients and contaminants; C) Regulatory, ethical, legal and social aspects of food biotechnology.
UNIT II: Practices in Food Biotechnology
2.1 A) Metabolic engineering of bacteria for food ingredients; B) Citric acid production; C) Microbial production of oils and fats.
2.2 A) Production of amino acids – physiological and genetic approaches; B) Production of pectinases – utilization in food processing; C) Microbial technology of food flavor production.
2.3 A) Genetic engineering of yeast; B) Food applications of algae– microalgae for food production; C) Enzymes for food applications.
2.4 A) Applications of transgenic plants in food production; B) Engineering starch, alternative sweeteners, plant oils for food use; C) Improving processing properties of crops.
2.5 A) Applications of transgenic animals in food production; B) Transgenic fish – improved fish growth rate; C) Egg as bioreactor.
UNIT III: Practices in Food Biotechnology
3.1 A) Food yeasts and derivatives – industrial processes; B) Alcoholic beverages – production processes; C) Bread and related products – ingredients and formulations, production processes.
3.2 A) Dairy products – manufacture of dairy products, metabolic systems in lactic acid bacteria, genetic modifications of lactic acid bacteria; B) Dairy products – applications of genetic engineering, designer milk, genetically modified cheese; C) Fermented meat products.
3.3 A) Fermented fish products; B) Fermented vegetable products – fermented soy products; C) Mushroom cultivation & preservation.
3.4 A) Production of vitamins, pigments; B) Microbial enzymes -overview, production of amylases, lipases, polygalacturonase; C) Microbial polysaccharides – overview, Xanthan gum production.
3.5 A) Functional foods – probiotics, prebiotics, synbiotics; B) Nutraceutical enrichment of food crops – minerals and vitamins; C) Food safety issues – safety evaluation of novel products (genetically modified plants, animals and microorganisms and their products), detection methods of GM crops, transgenic animals & fish, containment.
PRACTICALS: BT303P: FOOD BIOTECHNOLOGY
Unit - I
1. Qualitativeandquantitativeanalysisofcarbohydratesandproteins in food.
2. Preparation and evaluation of cheese or fermented product.
3. Determination of fat content in milk.
4. Estimation of vitamins- vitamin A, C and riboflavin.
5. Determination of aflatoxin in food.
6. Tests for pesticidal residues in food.
7. Determination of aflatoxin in food.
8. Enumeration and identification of microorganisms in probiotic foods.
REFERENCE BOOKS
1. Swaminathan M.S. Dr. Hand Book of Food and Nutrition
2. Sumati R. Mudambi and M. V, Rajgopal. Fundamentals of Food and Nutrition
3. Nutrient Requirements and Recommended Dietary Allowances for Indians. National Institute of Nutrition, Indian Council of Medical Research,2010
4. Aurand, L.W. and Woods, A.E.1973. Food Chemistry. AVI, Westport
5. Birch, G.G., Cameron, A.G. and Spencer, M. 1986. Food Science, 3rdEd. Pergamon Press, New York.
6. Rosenthal, I. 1991. Milk and Milk Products. VCH, New York
7. Warner, J. M. 1976. Principles of Dairy Processing. Wiley Eastern Ltd. New Delhi
8. Krammer, A. and Twigg, B.A. 1970. Quality Control for the Food Industry. 3 rd Edn. AVI, Westport
9. Pattee, H.E.Ed.1985.EvaluationofQualityofFruitsand Vegetables. AVI, Westport
10. Ranganna, S.1986. Handbook of Analysis and Quality Control for Fruits and Vegetable Products. Tata Mc Graw Hill, NewDelhi
11. Joshi,V.K.andPandey,A.Ed.1999.Biotechnology.FoodFermentation,( 2Vol.set).EducationPubl.NewDelhi
12. Knorr, D.1982. Food Biotechnology. Marcel Dekker, New York
13. Inteas Alli: Food Quality Assurance: Principles and practices, CRCPress LLC
14. Knechtes P.L.:FoodSafety: TheoryandPractice,JonesandBartlettLearning,USA 15. R.AGarg: The Food Safety and StandardAct,2006 along with Rules and regulation,(2011)Commercial Law Publisher(India)Pvt. Ltd
16. Food Biotechnology by Mason Sutton & Skylar Barr, published by Ed-Tech Press 2018
17. Enzymes in Food Biotechnology: Production, Applications, and Future Prospects edited by Mohammed Kuddus, published by Academic Press 2018.
18. Functional Foods and Biotechnology: Biotransformation and Analysis of Functional Foods and Ingredients edited by Kalidas Shetty, Dipayan Sarkar, published by CRC Press 2020
Paper IV A (ELECTIVE) BT 304 T (A) Animal Biotechnology
1. Course Objectives: (C.Obj):
a. To learn the technique of cell culture and its applications
b. Understand various classical and modern methods of animal improvement
c. To learn biotechniques involved in the creation of genetically modified organisms
d. To comprehend the importance and use of mouse as a disease model
2. Course Outcomes: (C.O):
a. Understanding the basics of cell culture technique and its utility in biotechnology
b. Comprehension of animal breeding methods and techniques
c. Acquaintance with modern techniques to create transgenic, knock-out and knock-in animals d. Knowledge regarding the use of mouse to model a disease and its utility in biotechnology
Course Plan/Schedule
Unit I 1. A) Cell culture technique: cell culture media and sterilization techniques and cryopreservation B) Immortalization and transformation; HeLa, A549, MCF7, K562 and HEK293 cell lines C) Cell maintenance and cell adaptation and contact inhibition
2. A) Characteristics of cells in culture and methods of separation of various cell types B) Anchorage dependence, stem cell culture and embryonic stem cell culture C) 3D cell culture, organoids as disease models
3. A) Manipulation of cells: cell transfection (electroporation and chemical methods) B) Transduction C) Synchronization of cell cultures
4. A) Production of secondary metabolites and scaling up of animal cell culture B) Tissue culture as a screening system and tissue engineering C) Mass production of biologically important compounds
5. A) Harvesting of recombinant products B) Purification and assaying of recombinant products C) Applications of cell culture in diagnostics and industry
Unit II 1. A) Conventional methods of animal Improvement- selectivebreeding, cross breeding B) Principles of animal breeding; structure of the livestock breeding industry: dairy cattle, beef cattle, swine, sheep, poultry and aqua culture C) Importance of animal improvement and its applications
2. A) Animal improvement techniques: Semen collection and artificial insemination B) Estrus synchronization and super ovulation C) In vitro maturation of oocytes andin vitro fertilization
3. A) Embryo collection and transfer B) Intracytoplasmic sperm injection C) Somatic cell nuclear transfer and embryo sexing
4. A) Identification and isolation of genes of economic importance B) Physical and genetic maps C) Candidate gene approach
5. A) Molecular markers and their application in animal improvement: RFLP and RAPD B) Microsatellite/minisatellite markers, SNP Markers and DNA fingerprinting C) Genetic tools and their applications: Southern blotting, Northern blotting and DNA sequencing
Unit III 1. A) Mouse as a disease model B) Breeding of mouse and maintaining the colonies C) Mouse strains and their utility
2. A) Generation of transgenic animals and their applications B) Creation of knock-out mouse and its applications C) Generation of knock-in mouse and its applications
3. A) Double transgenics, double knock-outs and their utility in deciphering biology B) Spontaneous and inducible gene expression, tissue-specific gene expression in mouse C) Humanized mouse and its applications
4. A) Genome editing tools: zinc finger nucleases (ZFNs), TALENs and CRISPR-Cas System B) Cancer models (carcinogen injection; bone marrow transplantation; xenografts) C) Cancer models (retrovirus- lentivirus- & adenovirus-based models)
5. A) Infectious disease models (bacterial, fungal and viral diseases) B) Metabolic disease models (Diabetes and obesity) C) Neurodegenerative disease models (Alzheimer’s, Huntington’s and Parkinson’s)
ANIMAL BIOTECHNOLOGY PRACTICALS
Unit I
1. Sterilization technique in mammalian cell culture
2. Freezing of cells
3.Thawing of frozen cells for culturing
4.Isolation of primary cells for culturing
5.Cell counting
6.Preparation of culture media
7.Culturing of suspension cells
8.Culturing of adherent cells
Unit II
1.Isolation of nucleic acids and proteins from cultured cells
2.Mammalian cell transfection (transient)
3.Testing of gene expression by SDS-PAGE
4.Testing of gene expression by western blotting
5.Testing of gene expression by fluorescence microscopy
6.Testing of gene expression by qRT-PCR
7.Testing of pharmacological activity of cancer drugs
REFERENCE BOOKS
1. Practical animal breeding. Blackwell Science.
2. Houdebine L.M. Animal transgenesis and cloning.Wiley Publishers.
3. R. Ian Freshney. Culture of animal cells: a manual of basic technique andspecialized applications.
4. Akano IE. DNA technology. IAP Academic Press.
5. Micklos DA, Fryer GA &Crotty DA. DNA science.Cold Spring Harbour.
6. Setlow JK. Genetic Engineering - Principles and methods. Springer.
7. Hare WCD & Elizabeth L Singh. Cytogenetics in animal reproduction.CABI.
8. Stine GJ. The new human genetics.Wm C Brown Publ.
9. Summer AT &Chandley AC. Chromosome today. Chapman & Hall.
10. Falconer DS & Mackay TFC. An introduction to quantitative genetics.Longman.
11. Jain JP. Statistical techniques in quantitative genetics. Tata McGraw-Hill.
12. Pirchner F. Population genetics in animal breeding. S. Chand.
13. Plumer. Practical biochemistry
14. Sambrook et al. Molecular cloning Volume 1, 2, 3.
15. Wilson K. and Walker J. Principles and techniques of biochemistry and molecularbiology
16. Harlow Ed and Lane D. Antibodies: a laboratory manual
17. Cell biology techniques: formulated by indian society of cell biology
18. Pasternack and Glick. Molecular biotechnology nsfection efficiency (using fluorescence and confocal microscopes)
1. Practical animal breeding. Blackwell Science.
2. Houdebine L.M. Animal transgenesis and cloning. Wiley Publishers.
3. R. Ian Freshney. Culture of animal cells: a manual of basic technique and specialized applications.
4. Akano IE. DNA technology. IAP Academic Press.
5. Micklos DA, Fryer GA & Crotty DA. DNA science. Cold Spring Harbour.
6. Setlow JK. Genetic Engineering - Principles and methods. Springer.
7. Hare WCD & Elizabeth L Singh. Cytogenetics in animal reproduction. CABI.
8. Stine GJ. The new human genetics. Wm C Brown Publ.
9. Summer AT & Chandley AC. Chromosome today. Chapman & Hall.
10. Falconer DS & Mackay TFC. An introduction to quantitative genetics. Longman.
11. Jain JP. Statistical techniques in quantitative genetics. Tata McGraw-Hill.
12. Pirchner F. Population genetics in animal breeding. S. Chand.
13. Plumer. Practical biochemistry
14. Sambrook et al. Molecular cloning Volume 1, 2, 3.
15. Wilson K. and Walker J. Principles and techniques of biochemistry and molecular biology
16. Harlow Ed and Lane D. Antibodies: a laboratory manual
17. Cell biology techniques: formulated by indian society of cell biology
18. Pasternack and Glick. Molecular biotechnology
Paper IV B (ELECTIVE) BT 304 T (B) Protein Engineering
1. Course Objectives: (C.Obj.):
a. To give an overview and impart conceptual understanding of protein structure.
b. To give molecular insights into protein folding pathways and describe the range of techniques developed over recent years to study proteins.
c. To highlight advancements in protein engineering and their application to study protein conformations.
d. To create a deeper understanding of the significance of prediction and design of protein structures using bioinformatics tools
2. Course Outcomes: (C.O):
a. Enables understanding and exploring protein characteristics that lay the foundation to protein engineering studies.
b. Acquire knowledge of protein folding mechanisms and familiarize with bioanalytical techniques.
c. Provide an advanced understanding of the core principles and applications of various important techniques employed for protein structure conformation studies.
d. Facilitate to carry out various in silico studies to build protein models and study protein ligand interactions that aid in drug design.
Course Plan/Schedule
UNIT 1: Protein structural families
1. Introduction: A) Basic structural principles: amino acids and their conformational accessibilities Amino acids: chemical properties, active site residues B) Dihedral angles propensity in the proteins, Ramachandran plot C) Protein Sequencing methods
2.Protein Families A) Motifs of protein structures and their packing; schematic and topology diagrams B) Protein interactions and their functions, applications C) Families of protein structures: alpha, alpha/beta, beta, small, etc
3. Chaperones and their role in protein folding A) Structure of chaperones and role of chaperones in protein folding B) Chaperone families C) Osmolyte assisted protein folding
4.Protein folding A) Protein folding pathways in prokaryotes B) Protein folding pathways in Eukaryotes C) Single and multiple folding pathways
5) Protein Kinetics A) Protein denaturation B) Renaturation of single domain proteins C) Renaturation of multidomain domain proteins
UNIT 2: Protein engineering
1. Bio-analytical techniques to study proteins A) Applications of UV-visible-Fluorimetry and CD to study proteins B) Applications of HPLC and LC-MS C) Applications of NMR, X-Ray diffraction & Cryo-EM to study protein conformations
2. Rational protein design and directed evolution A) Strategies for protein engineering B) Random and site-directed mutagenesis C) Mutagenesis using various PCR based strategies
3. Structural and cellular proteins and its role in nanotechnology A) Concept of proteome, proteomics B) DNA binding proteins C) Protein nanotechnology
4. Enzymology and refolding of proteins A) Role of low-fidelity enzymes in protein engineering B) Inclusion bodies C) Recovery of active proteins
5.Regulation of Protein engineering A) Phage display systems B) Expressional vectors C) Cell free translation system
UNIT 3: Prediction and design of protein structures
1 Evolution of protein A) Gene shuffling and B) directed evolution of proteins C) Protein backbone changes,
2.Protein databases and structural alignments A) Protein structure on the world wide web: different databases and their uses-PDB, SCOP, CATH B) Similar structure and function of homologous proteins C) Multiple structural alignment
3. Protein structure prediction & threading A) Homology method for protein structure prediction B) Ab-initio method for protein structure prediction C) Protein threading
4. Protein ligand interactions A) Rational protein design B) Ligand design and protein docking C) Structure based drug design and case studies
5. Approaches & applications of protein engineering A) Antibody engineering B) Enzyme engineering C) Protein Engineered Biomaterials D) Protein engineering and its application in Food industry E) Protein engineering in Environmental application
PROTEIN ENGINEERING PRACTICALS:
1. In-silico Site directed mutagenesis, energy minimizations and simulations
2. In-vitro site directed mutagenesis of enzymes by using PCR method
3. Over expression & optimization of targeted protein
4.Protein purification by using Ni-NTA affinity column chromatography
5. Protein purification by using Size exclusion column chromatography (AKTA)
6. Analysis of purified protein by electrophoresis
7. Analysis of purified protein by MALDI-TOF
8. Ligand-protein docking
REFERENCE BOOKS
1. Introduction to protein structure, Garland Press. Carl Branden and John Tooze, Structure and mechanism in protein science. Alan Fersht, Freeman
2. Protein engineering in industrial biotechnology, Academic Publishers. Ed. Lilia Alberghina, Harwood
3. Understanding Enzymes. T. Palmer, Prentice Hall 4. Modelling Biological Systems, Springer. Haefner