Genetic Engineering and rDNA Technology1.pdf

Uka Tarsadia University B.Sc. Microbiology BT5005: Genetic Engineering and rDNA Technology Semester - V EFFECTIVE FROM July-2021 Version 1.01  Uka Tarsadia University B.Sc....

Uka Tarsadia University B.Sc. Microbiology BT5005: Genetic Engineering and rDNA Technology Semester - V EFFECTIVE FROM July-2021 Version 1.01  Uka Tarsadia University B.Sc. Subject Hours Microbiology 5th Sem BT5005: Genetic Engineering and rDNA Technology 4hrs/week (Theory) 4 Credits Objectives:  The objective is to impart fundamental knowledge to students about theoretical aspects and applications of rDNA technology and genetic engineering for human benefits  To acquire adequate knowledge & necessary skills related to the different techniques involved in genetic engineering. Outcome: Upon completion of the course, the student shall be able to comprehend CO1 Players of gene cloning like DNA modifying enzymes, Cloning vector and Expression vector. CO2 Steps of gene cloning, library preparation and screening of positive clone from library. CO3 Advanced techniques of Recombinant DNA Technology CO4 Information about the few important recombinant proteins products and basics of Gene therapy techniques Sr. No. Topic Hours Unit – I 12 1 Introduction and Objectives of Gene Cloning Introduction o What is genetic engineering and recombinant DNA technology, Outline Steps of Gene Cloning MOLECULAR TOOLS FOR GENE CLONING: o Enzymes: Nucleases: Exonucleases and Endonucleases, Restriction Enzymes (Type I, Type II, Type III, Type IV & Type V), RNase, Methylases: CpG Methylase, Dam Methylase, Dcm Methylase, Polymerases: DNA Pol I, Klenow Fragments, Reverse Transcriptase, Taq & Pfu Polymerases, Ligases: T4 DNA Ligase, E.coli DNA Ligase, T4 RNA Ligase, End Modifying Enzymes: Terminal Transferase, T4 Polynucleotide Kinase, Alkaline Phosphatases Cloning Vectors Desirable properties of vectors Plasmid Vectors, Phage Vectors, Cosmids, Phagemids, BACs, Yeast Vectors (YACs),  Uka Tarsadia University Expression Vector: pET, pMAl, pGEX Unit – II 2  Strategies of Genetic Engineering, Gene Cloning In 12 Prokaryotes Isolation of DNA (Genome and Plasmid) to be cloned Construction of genomic library and cDNA Library. Isolation of mRNA, reverse transcriptase, oligo dC tailingAddition of oligo G primer Synthesis of second strand of cDNA Insertion of foreign DNA fragment into vector; linkers, adaptors and homopolymer tailing o Methods of gene transfer in prokaryotic and eukaryotic cells, o Transformation and growth of cells o Selection of Clones; Insertion inactivation and Blue White screening, colony hybridization techniques o Recombinant selection and screening methods:genetic, immunochemical, Expression of cloned DNA molecules and maximization of expression Unit – III 3  Advanced Techniques rDNA technology 12 Blotting Techniques: Southern, Northern & Western blotting, Autoradiography, Hybridization, Molecular Probes and Nucleic acid labeling, DNA sequencing, Polymerase Chain Reaction: PCR, Real Time PCR, RT PCR Mutagenesis, Analysis of gene expression, Methods for analysis of gene expression at RNA and protein level, large scale expression, such as micro array based techniques. Protein Engineering: Site Directed Mutagenesis. Oligonucleotide Directed Mutagenesis, Error prone PCR Protein-DNA interaction : EMSA, ChIP , Reporter Gene Assays Protein-Protein Interactions: MBP-Pull down assay, Coimmunoprecipitation assay, Yeast two hybrid assay etc. Unit – IV 4  APPLICATIONS OF GENETIC ENGINEERING 12 o Recombinant Products o Genetically Engineered Microorganisms, o Therapeutic proteins: Human Insulin, Interferon,  Uka Tarsadia University Human Growth hormone, Growth factors, Monoclonal Antibodies, Therapeutic oligonucleotides, Vaccines o Applications of gene cloning techniques in Agriculture, medical and environmental and forensic science o Gene Therapy. Sr. No. Practical 2 Credit 1 Isolation of genomic DNA from suitable source and quantification by DPA method and using Spectrophotometer. 2 PCR amplification and analysis by agarose gel electrophoresis. 3 Isolation of total RNA and quantification by Orcinol method and using UV Spectrophotometer. 4 Preparation of plasmid, pET-28a from E.coli DH5α, gel analysis and spectrophotometric analysis. 5 Restriction Digestion analysis of a given plasmid. 6 Preparation of competent cells. 7 Transformation in E.coli with recombinant vector. Reference books: 1. Biotechnology: The Biological Principles by M. D. Trevan, S. Boffey, K.H. Goulding and P. Stanburry TATA McGraw Hill Publishing Company Limited, New Delhi. 2. Biotechnology; Smith, Cambridge Press. 3. Advanced Molecular Biology; Twyman R.M. 4. Microbiology; Atlas R. M. 5. Microbiology- Prescott L.M. 6. Microbial Genetics; Freifilder D. 7. Principles of gene Manipulation; Old and Primrose. 8. Principles of Gene Manipulation and Genome; Primrose and Twyman (7 th Edition) Blackwell Publishing. 9. A textbook of Biotechnology by R.C. Dubey. 10. Textbook of Biotechnology by H K Das. 11. Biotechnology By B D Singh. 12. Gene cloning and DNA Analysis by T. A. Brown. Course objectives and Course outcomes mapping:  To develop a comprehensive understanding of different types of DNA modifying enzymes and the details of different cloning and expression vectors: CO1, CO2, CO3, CO4.  To acquire adequate knowledge of strategies in genetic engineering advanced techniques in recombinant DNA Technology and getting the insights of construction of recombinant proteins, gene therapy: CO1, CO2, CO3, CO4.  Uka Tarsadia University Course units and Course outcome mapping: CO1 CO2 CO3 CO4 Introduction of Gene cloning and players of gene √ cloning Strategies of Gene Cloning √ Advanced techniques of genetic engineering √ Recombinant Products and gene therapy √ Programme Outcomes PO 1: Knowledge: The students acquire strong theoretical background along with necessary skills in microbiological sciences and have the ability to use this knowledge in industry, hospitals, community and institutes or any other profession they would like to pursue. PO 2: Core Competence: Microbiology offers challenges to human intellect to find out the cause of deadly diseases in plants and animals at large and in humans in particular. At the same time, it also provides clues for finding out the remedy for diseases in the form of medicines. Microbiology plays an important role in developing many useful products for man and cleaning the environment. PO 3: Breadth: Microbiology, being a specialized branch of biology deals with the effect of microorganisms on other living organisms. The health of humans and existence of life on earth largely depend on microbial interactions. PO 4: Preparation: The curriculum is designed in such a way that in the first year the students are exposed to the fundamental aspects of plant science, animal science, microbial world and chemistry. Subsequently, they are made to learn specialized aspects such as enzymology, immunology, bacteriology, virology, pathology, genetic engineering etc along with practical applications. In addition, they are also introduced to the industrial aspects such as dairy technology, fermentation technology, enzyme technology and business entrepreneurial skills. During fourth and fifth year the students are also supposed to work on the research project and submit the findings in the form of dissertation. PEO 5: Professionalism: The career opportunities to choose from as a microbiologist are manifold. A post graduate degree in microbiology will qualify them for technical, research, and clinical positions. PEO 6: Evaluation: Academic performance evaluation of a student is according to a letter grading system = Total of Continuous Internal Evaluation (CIE) + Semester End Examination (SEE). Programme Outcomes and Course Outcomes mapping:  Uka Tarsadia University Programme Course outcomes Out come CO1 CO2 CO3 CO4 PO1 √ √ √ √ PO2 √ √ √ √ PO3 √ √ √ √ PO4 √ √ √ √ PO5 - - √ √ PO6 √ √ √ √