Skill Development

Skill Development under M.Tech. Marine Biotechnology Programme

Restructured Curriculum lays a strong Foundation in the Following Areas

  1. Marine Sciences
  2. Marine Microbiology
  3. Marine Genomics and Proteomics
  4. Marine Bioprospecting
  5. Computational and Systems Biology
  6. Aquatic Animal Health
  7. Brood Stock Development
  8. Bioprocess Technology
  9. Marine Algal Biotechnology
  10. Biostatistics

Areas of Specialization for Research Project

  1. Sustainable Marine Food Production Systems
  2. Marine Bio-Fuels
  3. Climate Change and Biodiversity Conservation
  4. Pharmaceuticals from Oceans

Skill Development

  1. Recombinant DNA Technology and Transgenesis
  2. Animal Cell Culture and Hybridoma Technology and
  3. Bioprocess Technology
  4. Fish Disease Diagnosis
  5. Preventive Health Care in Aquaculture.
  6. Therapy in fish health
  7. Brood stock development and Management
  8. Sustainable Aquaculture Systems
  9. Marine Bio-prospecting and Drug Discovery
  10. Marine Bio-fuel Technology

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LAB I- RECOMBINANT DNA TECHNOLOGY

1.Preparation of buffers and reagents for molecular biology.

2.RNA isolation and Northern blotting.

3.Primer design for gene expression experiments

4.cDNA synthesis, PCR amplification of selected genes, their cloning and expression.

5.Preparation of plasmid, pET -32a+ from E.coli DH5a.

6.Restriction digestion of vectors (gel analysis) and insert.

7.Vector and insert ligation, and transformation in E.coli DH5a

8.Plasmid isolation and confirming recombinant by PCR and RE digestion.

9.Transformation of recombinant plasmid in E. coli BL 21 (DE3) strain

10.Induction with IPTG and extraction of proteins

11.Protein analysis using SDS PAGE

12.Purification of protein on Ni - NTA column and analysis of the material by Western blotting to check purity.

13.2-dimensional Gel electrophoresis

14.NMR, MS -MS, X-ray crystallography (Demonstrations)

15.Quantification of genes using real time PCR

16.DNA sequencing methods (demonstration)

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LAB 2 - Hybridoma Technology

1.Antigen selection and purification

2.Immunization of mice

3.Hybridization with myeloma cells

4.Selection of hybrid clones- HAT system

5.Clonal selection

6.Screening and sub-cloning of hybridomas

7.Production of monoclonal antibodies

8.Checking cross reactivity.

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LAB 3 - MARINE MICROBIAL DIVERSITY

  1. Isolation of marine bacteria – heterotrophs (organotrophs), photoautotrophs, chemolithotroph,; purification; characterization – phenotypic and genotypic; identification.
  2. Isolation of marine obligate bacteria - purification; characterization – phenotypic and genotypic; identification.
  3. Isolation of marine fungi - purification; characterization – phenotypic and genotypic; identification.
  4. Isolation of marine microalgae - purification; characterization – phenotypic and genotypic; identification.
  5. Isolation of marine protozoans - purification; characterization – phenotypic and genotypic; identification.
  6. Isolation of marine viruses - purification; characterization – phenotypic and genotypic; identification.
  7. Quantification of marine micoorganisms- bacteria, fungi, protozoans, microalgae and viruses.

Molecular methods in marine microbial diversity

  1. DNA extraction and quantification from prokaryotes, eukaryotes and marine environmental samples.
  2. Agarose gel electrophoresis and purification of DNA from gel.
  3. Pulsed Field Gel Electrophoresis for high molecular weight metagenomic DNA isolation
  4. DNA restriction analysis and Southern blotting.
  5. PCR amplification of 16S, 18S rRNA gene and internal transcribed spacers, purification of the PCR products.
  6. Ligation, transformation and screening of transformants by colony PCR.
  7. Plasmid extraction, restriction digestion of the plasmid DNA.
  8. Bacterial 16S rRNA and eukaryotic 18S rRNA genes and internal transcribed spacers sequencing and sequence analysis.

DNA Fingerprinting Techniques

  1. RFLP/RAPD/AFLP/ Microsatellite.
  2. DNA Barcoding.
  3. Denaturing Gradient Gel Electrophoresis.
  4. Fluorescent In-Situ Hybridization
  5. Lipid profiling and species identification (demonstration)

Bioinformatics for microbial diversity analyses

  1. Primer/probe design for PCR
  2. Data retrieval tools: Entrez, SRS, Literature search: Pubmed, Medline, OMIM
  3. Sequence alignment: DOT MATRIX approach
  4. Protein and nucleic acid alignment programs.
  5. Statistical evaluation of sequence alignment scores.
  6. Multiple sequence alignment.
  7. Generation of phylogenetic tree, Phylogenetic analysis.
  8. Microbial diversity analyses

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LAB 4 BIOTECHNOLOGICAL INTERVENTIONS IN AQUATIC ANIMAL HEALTH

  1. Disease Diagnosis and pathogen detection
    1. Fish sampling for laboratory observation.
    2. Anatomy of fish and shellfish: Dissections to study the internal organs of fishes and shellfishes.
    3. Clinical study – skin, internal organs and gill biopsy.
    4. Histological methods.
    5. Microbiological methods, isolation of aetiological agents - identification, characterization.
    6. Immunodiagnostic techniques – agglutination, haemagglutination, precipitation, latex agglutination, immuno fluorescence, immunofluorescence histochemistry, radial immuno diffusion, immuno electrophoresis, counter current electrophoresis and ELISA.
    7. DNA based diagnostic techniques: Shrimp and fish pathogens.
    8. Use of cell lines in disease diagnosis.
    9. Electron microscopy in disease diagnosis (Demonstration)
    10. Koch’s postulates.
  2. Disease Management protocol
    1. Prophylaxis - Environmental management.
    2. Bioremediation - application of probiotics, vaccines.
    3. Therapy – Application of antibiotics in non-edible fish.
  3. Fish and Shell fish immunology
    1. Determination of total and differential haemocyte/blood count.
    2. Estimation of protein.
    3. Prophenol oxidase activity.
    4. NBT reduction assay.
    5. Alkaline phosphatase and Acid phosphatase.
    6. Lysozyme activity.
    7. Superoxide dismutase, Catalase , Glutathione peroxidase assay.
    8. Haemeagglutination.
    9. Detection and quantification of antibodies in serum.
  4. Cell line development and maintenance :
    1. Preparation of media.
    2. Developing primary cell culture.
    3. Sub culturing and cell line maintenance – anchor dependent and suspension cell cultures.
    4. Enumeration of viable cells by dye up take and exclusion.
    5. Split ratio and growth cycle. 
    6. MTT assay.
    7. BrdU assay.
    8. Cytoskeleton staining.
    9. Cryo preservation.
  5. Application of cell lines :
    1. Virus isolation – plaque assay, Virus titration - TCID 50.
    2. Detection of viruses through immunohistochemistry/immunoflourescent staining and  toxicological studies – LD 50.

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LAB 5 BIOPROPECTING AND BIOPROCESS ENGINEERING

A. Bioprospecting

  1. Collection and identification of marine organisms.
  2. Extraction of bioactive compounds with different solvent systems.
  3. Screening of extracts for anticancer activity
    1. Brine shrimp lethality assay
    2. Sulforhodamine B assay/MTT assay
    3. Annexin V/FITP assay
    4. TUNEL Assay
  4. Screening for Anti-bacterial/antifungal and antiviral activity of extract/compound
  5. Screening of anti-inflammatory activity of compound by Griess assay for quantification of NO on RAW 264.7 cell line
  6. Isolation and partial characterization of lead compound by thin layer chromatography.
  7. Separation and Purification of natural product by column chromatography and HPLC.
  8. Gene prediction.
  9. Structure analysis and drug design: protein structure prediction tools.
  10. Structure visualization tools-Swiss PdbViewer, RASMOL, Cn3D.
  11. Docking tools - ArgusLab, AutoDock
  12. Tools for ADME-Tox prediction
  13. MOE

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B. Bioprocess Engineering

  1. Designing and optimization of culture media by Response Surface Methodology (RSM).
  2. Downstream processing for bioactive compounds and microbial cells.
  3. Submerged fermentation and Solid state fermentation.
  4. Determination of kinetics of fermentation.
  5. Batch growth kinetics.
  6. Fed-batch culture.
  7. Measurement of KLa.
  8. Mixing time.
  9. Residence time distribution.
  10. Enzyme kinetics with and without inhibitor.
  11. Immobilized enzyme kinetics.
  12. Protein precipitation and fractionation.
  13. Ion-exchange chromatography.
  14. Size exclusion chromatography.

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LAB 6 HIGH HEALTH BROOD STOCK DEVELOPMENT

PART A - Marker assisted selection

1.Molecular markers - designing,amplification and sequencing

2.Marker assisted selection

PART B - Fish Transgenesis

1.Gene selection

2.Amplification

3.Sequencing

4.Generation of gene construct

5.Transformation

6.Examination of the phenotype and genotype.

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