Domain Track: Genetic Engineering and Genomics

Teacher

Dr Rukmini Mishra

Category

Domain Courses

Course Attendees

Still no participant

Course Reviews

Still no reviews

Domain Track Title: Genetic Engineering and Genomics

 

Track Total Credit (T-P-P): 3-15-11 :

 

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. Plant Molecular Biology 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 silico identification of pharmacophore for plant diseases.
3.In vitro micro-propagation of banana and hardening.
4.Commercial production of horticultural crops.
5.Standardization of protocol for in vitro micro-propagation of horticulture crops.
6.DNA isolation, amplification and gel electrophoresis of plant sample.
7.Phenotyping and genotyping of rice varieties for abiotic stress tolerance.
8.Genome editing techniques towards crop improvement.

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: Gene Information Resources:
NCBI GenBank resources
Protein Information Resource (PIR): SWISS PROT and Protein Data Bank (PDB)
Other specialized genomic resources.
DNA sequence analysis: cDNA libraries and Expressed Sequence Tags (ESTs), EST analysis, Pairwise alignment techniques, Multiple sequence alignment.
Python Tools for Computational Molecular Biology: Biopython - Bio.AlignIO - alignment input/output, Bio.SeqIO - sequence input/output, Bio.PopGen - population genetics, Bio.PDB - structural bioinformatics, Biopython’s BioSQL interface.
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), Retrival 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:
Recombinant DNA technology: Genomic DNA & plasmid DNA isolation and purification, construction of recombinant DNA and expression cassettes, Transformation (mobilization of vectors into competent bacteria), selection and analysis of recombinant clones, genomic DNA and cDNA 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, optimized protocols for Agrobacterium-mediated genetic transformation, physical and chemical methods of gene transfer. Direct gene transfer methods (particle bombardment/ micro projectile / biolistic, electroporation, microinjection), chemical methods (PEG - mediated, calcium phosphate co-precipitation).
Genome Editing: 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. Plant Molecular Biology
Theory:
Hormone regulatory pathways:
Ethylene, Cytokinin, Auxin and ABA, SA and JA; ABC Model of Floral Development, Molecular basis of self-incompatibility, Regulation of flowering: photoperiod, vernalization, circadian rhythms and their gene responsiveness.
Molecular biology of abiotic stress responses: Cold, high temperature, sub-mergence, salinity and drought; Molecular Biology of plant-pathogen interactions.
Genome Mapping: Introduction to genomics, Forward and Reverse Genetic Approaches.
Genome sequencing projects: Human Genome Project, Genome sequencing projects for microbes, Genome sequencing projects for plants and animals, Accessing and retrieving genome project information from the web
Proteomics: Introduction to proteomics, Proteomics technologies: 2D-PAGE, isoelectric focusing, Mass spectrometry, MALDI-TOF, Yeast 2-hybrid system, Proteome databases.
Practice in Project mode: Demonstration of 2D-PAGE gel, 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

Session 1.7

Theory:
Methods of in silico Drug Design: structure and ligand based drug design approach

Practice:

Structure based drug design

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:

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. Plant Molecular Biology

Session 3.1
Theory:
Hormone Regulatory Pathways
Practice:

Effect of growth regulators on field crops (Phenotyping)

Session 3.2
Theory:
Regulation of flowering, photoperiod

Practice:

Effect of abiotic stress on field crops (Phenotyping)

Session 3.3
Theory:
Molecular analysis of abiotic stress tolerance among field crops

Practice:

Stress tolerance genes and their responsiveness (Genotyping)

Session 3.4
Theory:
Molecular analysis of biotic stress tolerance among field crops

Practice:

Molecular markers and their analysis

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: 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

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
Factors affecting micropropagation

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
Quantitative gene expression through qRT-PCR.

https://www.youtube.com/watch?v=sZbSMwEvVR0

Practice 2
In silico mapping of genomes.

Practice 3
Phylogenetic analysis

Practice 4
Genome sequencing, sequence assembly with an example of model organism Arabidopsis thaliana, etc.

Practice 4
Functional genomics: DNA chips and their use in transcriptome analysis

List of Projects to be done under the Domain

1.Exploration of available various sequences from different crops.
2.In silico identification of pharmacophore for plant diseases.
3.In vitro micro-propagation of banana and hardening.
4.Commercial production of horticultural crops.
5.Standardization of protocol for in vitro micro-propagation of horticulture crops.
6.DNA isolation, amplification and gel electrophoresis of plant sample.
7.Phenotyping and genotyping of rice varieties for abiotic stress tolerance.
8. Genome editing techniques towards crop improvement.

Our Main Teachers

Dr Rukmini Mishra

Assistant Professor
VIEW PROFILE

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 […]