Domain Track: Genetic Engineering and Genomics

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Dr Rukmini Mishra

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Domain Track Title: Genetic Engineering and Genomics

 

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:

  1. 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

https://www.youtube.com/watch?v=Phxkg5H5Q6E

Session 1.2

Theory:
Biological Databases, Classification and different types of Biological Database

Practice:

Demonstration and usage of protein databases: PDB, PIR, SwissProt, tREMBL

https://www.youtube.com/watch?v=VwNcfxBL4J0

Session 1.3

Theory:
DNA and Protein sequence analysis, Pairwise and multiple sequence alignment

Practice:

Sequence similarity serach using BLAST

https://www.youtube.com/watch?v=RzC-V67z5LA

Session 1.4

Theory:
Protein structural analysis, protein structure modeling and prediction

Practice:

Multiple sequence alignment using ClustalW

https://www.youtube.com/watch?v=TZaA_-4j19w

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

Molecular docking PPT

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

https://www.youtube.com/watch?v=r66YCsA4YqM

Session 1.9

Theory:
Create a simple sequence in Biopython for DNA, RNA and Protein Alphabets

Practice:

Create a simple sequence in Biopython

Session 1.10

Theory:
Sequence Alignment Tools in Biopython

Practice:

Sequence alignment using Biopython

https://www.youtube.com/watch?v=MxZCwiKusdE

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

https://www.youtube.com/watch?v=KRpik9mNRm0&t=8s

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)

Effect of abiotic stress on field crops Case Studies

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)

Molecular markers and their analysis GPL

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

RNA isolation from plants GPL

https://www.youtube.com/watch?v=k06zjuBpQT0

Session 3.9
Theory:
Transcriptome analysis for identification and functional annotation of gene

Practice:

Protein extraction from plants

Protein extraction from plants GPL Protocol

https://www.youtube.com/watch?v=ANy7XBmSolU

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

https://www.youtube.com/watch?v=0Y4dCVrl37k

Practice 2
Anther, Pollen and ovule culture, Embryo culture

Practice 3
Somatic embryogenesis – Induction and Development method

https://www.youtube.com/watch?v=GoZP-LQ4jcE

Practice 4
Protoplast isolation – Enzymatic and Mechanical method

https://www.youtube.com/watch?v=LjqmE3mnrQU

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.

https://www.youtube.com/watch?v=W5PsDSdudrI

Practice 2
DNA amplification by using PCR and optimization of factors affecting PCR

https://www.youtube.com/watch?v=iQsu3Kz9NYo

https://www.youtube.com/watch?v=matsiHSuoOw

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

Assistant Professor
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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 […]