Domain Track Title: Genetic Engineering and Genomics
Domain Track: Genetic Engineering and Genomics
Teacher
Dr Rukmini Mishra
Category
Domain Courses
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Track Total Credit (T-P-P): 9-9-11 :
Course Division (list of all divisions) :
Track Courses (3-6-0)
1. Computational Biology 1-2-0
2. Genetic Engineering and its applications 1-2-0
3. Genetics and Genomics 1-2-0
Practice Courses (0-9-0)
1. Molecular Genomics 0-3-0
2. Plant Tissue Culture Technologies 0-3-0
3. Techniques in Molecular Biology 0-3-0
AELP Project (0-0-11)
1.Exploration of available various sequences from different crops.
2.In vitro micro-propagation of banana and hardening.
3.Commercial production of horticultural crops.
4.Standardization of protocol for in vitro micro-propagation of horticulture crops.
5.DNA isolation, amplification and gel electrophoresis of plant sample.
6.Phenotyping and genotyping of rice varieties for abiotic stress tolerance.
7.Genome editing techniques towards crop improvement.
8. The RNA secondary structure variation in viruses and the possible implication in pathogenicity
9. Understanding RNA Protein interactions in viruses
Domain Track Objectives:
To impart basic knowledge of bioinformatics in in silico and genomics to the students studying agriculture.
Domain Track Learning Outcome:
• To get introduced to the basic concepts of Bioinformatics and its significance in Biological data analysis.
• To familiarize the students with recent tools used for genome analysis and their applications.
• To provide and train the students for commercial plant tissue culture raised plant production and meet the industrial requirement.
• To provide hands on training on basic molecular biology techniques.
Domain Syllabus:
- Computational Biology
Theory:
Biological databases: Overview of biological databases, nucleic acid & protein databases, primary, secondary, functional, composite, structural classification of databases. Alignment using BLAST and CLUSTAL W.
RNA and DNA folding: Introduction to RNA and DNA folding, Softwares and webservers used for DNA or RNA folding: Mfold, RNAfold, RNA structure
Insilico Drug Design: Basic Concepts, importance and application. Molecular Docking
Python Tools for Computational Molecular Biology: Introduction to Biopython
Practice in Project Mode: NCBI GenBank resources - Overview, Access, Data Usage, Confidentiality, International Nucleotide Sequence Database Collaboration, Data Type, European Nucleotide Archive - sequence assembly information and functional annotation, DNA Data Bank of Japan (DDBJ). Kyoto Encyclopedia of Genes and Genomes (KEGG), Retrieval of sequence/structure from databases, Visualization of structures, Docking of ligand receptors, BLAST exercises, Molecular models, drug design, Python based computational biology - Biopython.
Suggested Readings:
• Attwood TK & Parry-Smith DJ. 2003. Introduction to Bioinformatics. Pearson Education.
• Rastogi SC, Mendiratta N & Rastogi P. 2004. Bioinformatics: Concepts, Skills and Applications. CBS.
2. Genetic Engineering and its applications
Theory:
Plant Tissue culture: Cell culture media and sterilization techniques, Callus culture, nodal and tip culture, Protoplast and Embryo culture, Embryo culture and embryo rescue, protoplast isolation, culture and plant regeneration Somatic embryogenesis, Somaclonal variation and crop improvement, Germplasm conservation.
Recombinant DNA technology: Introduction to genetic engineering, restriction endonucleases and methylases; DNA ligase, T4 DNA polymerase, polynucleotide kinase, alkaline phosphatase. Gene cloning vectors: Plasmids; Bacteriophages; phagemids; Lambda vectors; Cosmids; Artificial chromosome vectors (YACs; BACs).Construction of recombinant DNA and expression cassettes, Transformation (mobilization of vectors into competent bacteria), selection and analysis of recombinant clones, Isolation of mRNA and total RNA; reverse transcriptase and cDNA synthesis; Genomic DNA & plasmid DNA isolation and purification, genomic DNA and cDNA libraries, screening of libraries.
Gene transfer in Plants: Vector mediated Gene transfer, Molecular basis of crown gall and hairy root diseases, features of Ti and Ri plasmids, mechanism of T-DNA transfer, role of virulence genes, vectors based on PTi & PRi, binary and co-integrate vectors, physical and chemical methods of gene transfer. Introduction to gene editing, ZFNs & TALENs, CRISPR/Cas System, Case studies of gene editing for crop improvement.
Practice in Project Mode: DNA isolation and purification, Callus induction, Agrobacterium mediated gene transfer in plants, vector construction, cloning and transformation.
Suggested Readings:
Glick, B. R. and Pasternak (2003). Molecular Biotechnology: Principles and Applications of Recombinant DNA. ASM Press, Washington, D. C., USA.
Kyte, L. and Kleyn, J. (1996). Plants from Test Tube to: an Introduction to Micro propagation, 3rd Ed. Timber press, Port land, USA.
Reference Book
Pollard, W. J. and Walker (1990).Plant Cell and Tissue Culture Vol VI. Humana press Clifton, USA.
3. Genetics and Genomics
Theory:
Basic of genetics: Mendel’s Laws, concept of dominance, segregation, independent assortment; Chromosome theory of inheritance.
Genome Mapping and Sequencing: Genetic and physical mapping, Markers for genetic mapping, Human Genome Project, Genome sequencing projects for microbes, Genome sequencing projects for plants and animals, Accessing and retrieving genome project information from the web. Genome sequencing: assembly, alignment and annotation. DNA sequencing strategies, Next generation sequencing strategies.
Functional genomics and proteomics: Introduction to genomics, transcriptome analysis for identification and functional annotation of gene, forward and reverse genetic approaches. Introduction to proteomics, proteomics technologies: 2D-PAGE, isoelectric focusing, Mass spectrometry.
Practice in Project mode: Demonstration of web tools for genome analysis. Effect of abiotic stress on field crops (Phenotyping), Effect of growth regulators on field crops (Phenotyping), Salinity/Cold/High Temperature stress tolerance genes and their responsiveness (Genotyping), Molecular markers (RAPD, SSR, AFLP etc) and their analysis.
Suggested Readings:
•Practical Applications of Plant Molecular Biology By Robert J. Henry.
• Biochemistry and Molecular Biology of Plants edited by Bob B. Buchanan, Wilhelm Gruissem, Russell L. Jones.
Session Plan
1. Computational Biology
Session 1.1
Theory:
Computational Biology: Basic Concepts, importance and application
Practice:
Demonstration and usage of web resources: EMBL, GenBank, ENTREZ
Session 1.2
Theory:
Biological Databases, Classification and different types of Biological Database
Practice:
Demonstration and usage of protein databases: PDB, PIR, SwissProt, tREMBL
Session 1.3
Theory:
DNA and Protein sequence analysis, Pairwise and multiple sequence alignment
Practice:
Sequence similarity serach using BLAST
Session 1.4
Theory:
Protein structural analysis, protein structure modeling and prediction
Practice:
Multiple sequence alignment using ClustalW
Session 1.5
Theory:
Molecular evolution, Phylogenetics analysis, analysis packages dealing with molecular evolution and phylogenesis
Practice:
Phylogenetic analysis through Neighbor joining method using MEGA tool
https://www.youtube.com/watch?v=6_XMKmFQ_w8
https://www.youtube.com/watch?v=encRU80nOHg
Session 1.6
Theory:
In silico Drug Design: Basic Concepts, importance and application
Practice:
Molecular docking
Session 1.7
Theory:
Methods of in silico Drug Design: structure and ligand based drug design approach
Practice:
Structure based drug design
Structure based drug design Material
Session 1.8
Theory:
Biopython: Introduction, important features and application of biopython in computational biology
Practice:
Demonstration of Biopython
Session 1.9
Theory:
Create a simple sequence in Biopython for DNA, RNA and Protein Alphabets
Practice:
Session 1.10
Theory:
Sequence Alignment Tools in Biopython
Practice:
Sequence alignment using Biopython
2. Genetic Engineering and its applications (1+2)
Session 2.1
Theory:
Plasmids, vectors and enzymes used in genetic engineering
Practice:
Restriction digestion
https://www.youtube.com/watch?v=GNMJBMtKKWU
https://www.youtube.com/watch?v=Ik_Pxht1LM0
Session 2.2
Theory:
Genomic DNA and plasmid DNA isolation and purification
Practice:
Isolation and purification of Plant DNA
https://www.youtube.com/watch?v=W5PsDSdudrI
https://www.youtube.com/watch?v=ukZp6Adu534&t=32s
Session 2.3
Theory:
Vecor construction, transformation and selection of transformants
Practice:
Plasmid DNA isolation and purification
Session 2.4
Theory:
Genomic library and cDNA library, screening of libraries.
Practice:
Quantitative and qualitative estimation of DNA/RNA
https://www.youtube.com/watch?v=VvusmIcA6i0&t=10s
Session 2.5
Theory:
Plant Transformation-Ti and Ri plasmids
Practice:
Elution of specific DNA fragment from PCR products
https://www.youtube.com/watch?v=yesNHd9h8k0
https://www.youtube.com/watch?v=K1ZyzvsHhOE
Session 2.6
Theory:
Molecular basis of crown gall disease and hairy root disease
Practice:
Cloning of DNA fragments into suitable vectors
https://www.youtube.com/watch?v=wTO-KmpZQgQ
Session 2.7
Theory:
Binary and Co-integrative vectors, Mechanism of DNA transfer
Practice:
Transformation of recombinant vectors into E. coli cells
https://www.youtube.com/watch?v=1rSWOSt3wVQ&t=163s
https://www.youtube.com/watch?v=wTO-KmpZQgQ&t=41s
Session 2.8
Theory:
Physical and Chemical methods of Gene transfer
Practice:
Selection and screening of positive transformants using colony PCR
https://www.youtube.com/watch?v=VqklR_8YRfA&t=29s
https://www.youtube.com/watch?v=ulA8xsVji80
https://www.youtube.com/watch?v=7Ul9RVYG5CM&t=4s
Session 2.9
Theory:
Introduction to gene editing, ZFNs and TALENs
Practice:
Demonstration of Agrobacterium infection in tobacco plants
https://www.youtube.com/watch?v=I5_2c52OPFw
https://www.youtube.com/watch?v=TvySiie0KEA
https://www.youtube.com/watch?v=XgeKbHf5MJk
Session 2.10
Theory:
CRISPR/Cas genome editing, case studies of editing tools in crop improvement
Practice:
Designing of sgRNAs using the RGEN tool
https://www.youtube.com/watch?v=2pp17E4E-O8
https://www.youtube.com/watch?v=4YKFw2KZA5o
3. Genetics and Genomics
Session 3.1
Theory:
Mendelian Genetics
Practice:
Karyotyping using photographs
Session 3.2
Theory:
Effect of abiotic stresses on field crops
Practice:
Effect of abiotic stress on field crops (Phenotyping)
Session 3.3
Theory:
Effect of biotic stress on among field crops
Practice:
Effect of biotic stress on field crops (Phenotyping)
Session 3.4
Theory:
Molecular analysis of biotic stress tolerance among field crops
Practice:
Molecular markers and their analysis (Genotyping)
Session 3.5
Theory:
Introduction to Genomics
Practice:
Demonstration of the working conditions of PCR
https://www.youtube.com/watch?v=mmgIClg0Y1k
https://www.youtube.com/watch?v=iQsu3Kz9NYo
Session 3.6
Theory:
DNA Sequencing, Genome sequencing projects for microbes, plants and animals
Practice:
Demonstration of the working conditions of qRTPCR
https://www.youtube.com/watch?v=4TF7VC4-4nQ
https://www.youtube.com/watch?v=pNY7znelCN0
Session 3.7
Theory:
Accessing and retrieving genome project information in in silico
Practice:
Retrieval of data from the genome sequences databases
https://www.youtube.com/watch?v=5teoROLvijg
https://www.youtube.com/watch?v=rDhElW5ox6w
Session 3.8
Theory:
Proteomics technologies: Protein Extraction, 2D-PAGE, isoelectric focusing, Mass spectrometry
Practice:
RNA isolation from plants
Session 3.9
Theory:
Transcriptome analysis for identification and functional annotation of gene
Practice:
Protein extraction from plants
Protein extraction from plants GPL Protocol
Session 3.10
Theory:
Gene function- forward and reverse genetics
Practice:
Synthesis of cDNA from supplied RNA using reverse transcription approach
https://www.youtube.com/watch?v=Paf5WRMizzQ
Practice Courses
4. Plant Tissue Culture Techniques (0-3-0) Practice-Session Plan
Practice 1
Micropropagation in plants
Practice 2
Anther, Pollen and ovule culture, Embryo culture
Practice 3
Somatic embryogenesis – Induction and Development method
Practice 4
Protoplast isolation – Enzymatic and Mechanical method
Practice 5
Vector-mediated gene transfer by using Agrobacterium tumefaciens
https://www.youtube.com/watch?v=NXNFR4cj68U&t=152s
https://www.youtube.com/watch?v=wTO-KmpZQgQ
5. Techniques in Molecular Biology (0-3-0) Practice-Session Plan
Practice 1
Isolation and purification of DNA from plant material.
Practice 2
DNA amplification by using PCR and optimization of factors affecting PCR
Practice 3
Gel electrophoresis- agarose and PAGE (nucleic acids and proteins).
https://www.youtube.com/watch?v=QEG8dz7cbnY&t=40s
https://www.youtube.com/watch?v=6mQGNDnOyH8
https://www.youtube.com/watch?v=pnBZeL8nFEo
Practice 4
Restriction digestion of plasmid and phage DNA
https://www.youtube.com/watch?v=5aHdSJW3NyE
https://www.youtube.com/watch?v=GsWo8dCivWs
Practice 5
Gene cloning – Recombinant DNA construction, transformation and selection of transformants
https://www.youtube.com/watch?v=sjwNtQYLKeU&t=74s
https://www.youtube.com/watch?v=3YRjpiBqSU4&t=25s
6. Molecular genomics (0-3-0) Practice-Session Plan
Practice 1
Demonstration of genome browsers- ENSEMBL, VISTA and UCSC Genome Browser
Practice 2
Identification of gene characteristics using ExPaSy tools
Practice 3
Phylogenetic analysis using genomic tools
Practice 4
Demonstration of genome assembly & alignment using web tools (for ex. Splign etc.)
Practice 5
Demonstration of genome comparison using MOAV tool or other web tools
List of Projects to be done under the Domain
1.Exploration of available various sequences from different crops.
2.In vitro micro-propagation of banana and hardening.
3.Commercial production of horticultural crops.
4.Standardization of protocol for in vitro micro-propagation of horticulture crops.
5.DNA isolation, amplification and gel electrophoresis of plant sample.
6.Phenotyping and genotyping of rice varieties for abiotic stress tolerance.
7. Genome editing techniques towards crop improvement.
8.NGS data analysis and data representation using open source tools or JMP software.
9.Understanding RNA-protein interactions in viruses
Our Main Teachers
Dr Rukmini Mishra is presently working as an Assistant Professor (Botany), School of Applied Science, Centurion University of Technology and Management. She has a PhD in Agricultural Biotechnology from ICAR-National Rice Research Institute, India. After PhD, she received the prestigious Young Scientist Award from the Dept. of Science & Technology, SERB, Govt. of India to […]