MSc Biotechnology - BT521 - Molecular Biology and Molecular Genetics


23 oct. 2013 (il y a 4 années et 11 mois)

233 vue(s)

MSc Biotechnology


Biology and Molecular Genetics

Credits: 5

1. What is Molecular Biology?

DNA as the hereditory material. RNA can also be the hereditory material, but only in some

Determination of radioactivity.

2. Size of
natural DNA molecule.

Estimation of DNA

Difference between DNA and RNA. Presence of phosphodiester linkage in RNA and DNA.
5' to 3' linkage.

Double strandedness of cellular DNA.

3. Antiparallel nature of the two strands of DNA.

4. Denaturation and renatura
tion of DNA. Tm. GC content from Tm. Renaturation kinetics of
DNA and complexity of DNA. Cot curves.

5. Separation of and determination of single stranded and double stranded DNA.

DNA hybridization
relatedness of difference genes and species.

6. Restri
ction endonuclease

an essential tool for gene cloning. DNA ligases. Plasmids.
Antibiotic resistance markers.

Transformation of E. coli.

7. Desirable characteristics of a plasmid cloning vector.

8. Centrifugation

velocity and equilibrium.

Messelson & St
ahl experiment.

9. Replication fork

John Cains experiment. Huberman & Riggs experiment on replication of
Chinese Hamster DNA. Inman experiment on replication of lambda DNA.

10. Genetic mapping

recombination frequency in bacteria and viruses


interrupted mating. Taylor map. Hfr strains.

11. Terminal redundancy and Circular permutation in T even phages.

12. DNA content in E. coli. Dichotomous replication.

13. Synchronization of E. coli cells.

14. Bi
directional replication in E. coli.

15. M
echanism of genome transfer during conjugation

Between Hfr and F strains of E. coli.

16. Mechanism of Genetic recombination.

17. Kornberg DNA polymerase.

18. Exonuclease II and exonuclease VI as integral part of Kornberg DNA polymerase.

19. Okazaki fragmen

20. The Pol AI mutant. DNA pol II and pol III. Kornberg's reappraisal of his old work.
Properties of pol I, pol II and pol III. Phenotyphic defects of pol A mutant. Ts mutants of
DNA replication and genetic mapping of the mutant loci.

21. Conversion of

single stranded circular DNA viruses into double stranded RF form.
Synthesis of viral plus strand from RF DNA.

22. OX gene A protein and its cleavage site. Rolling circle mode of synthesis. Involvement of
SSB protein.

23. Overlapping genes.

24. Insertion



25. Genome organization of prokaryotes

Viral genomes : General information about different types of viral genomes. Structural
organization of Retroviral genomes.

26. Transcription :

i. Flow of information from DNA to protein.
Organization of genes in bacteria. Colinearity of
genes and proteins. Operon concept.

ii. Process of transcription :

RNA polymerase subunit structure and function role of sigma factor in differential expression
of genes in bacteria.

Transcription units and

Cis elements. Promoter :

Consensus sequences affecting the promoter function. Constitutive and inducible promoters.
Operator sequences as regulatory cis sequences.

Initiation : Interaction of polymerase with the promoter and control at initiation. Attenua
Elongation. Termination : Rho dependent and Rho independent termination. Control at
termination : Attnuation. Antitermination.

Processing of primary transcripts in prokarytoes :

Processing of tRNA and rRNA. Cleavage of T7 early mRNAs by RNase III. C
ontrol at the
processing level.

iii. Regulation of transcription in bacteria : Introduction and repression. Repressor as a
regulatory molecule. Coordinated control of gene clusters. Positive and negative regulation :
Regulation of transcription of lac, trp
, ara, his, and gal operons. Regulation through catabolite
repression. CAP protein as a positive control factor.

iv. Transcriptional regulation in bacteriopohage Lambda. Lytic and Lysogenic switch. Role
of various regulatory proteins.

v. Sequential express
ion of genes in Bacteriophage T4, 2 T7, Mu, x 174 : Genome
organization and infection. Lectures

27. Translation :

Genetic code and codon bias. Essential components of translation. Ribosome : the site for
translation,sub unit composition and assembly. Role
of ribosomal RNA in translation. tRNA :
Salient features of tRNA. Aminoacyl Trna synthetases. Difference between initiator fmett
RNA and mettRNA, Suppressor tRNAs, frameshift suppression.Codon Anticodon
recognition :Wobble hypothesis. Process of translatio
n : Activation, Initiagtion, elongation
translocation and termination. Factors involved in various
steps.Peptidyltransferases.Transport of bacterial proteins : Cotranslational and Post
translational mechanisms. Control of geneexpression at translational le

MSc Biotechnology



Credits: 3

Growth Rate Analysis

Growth rate parameters : Specific growth rate, doubling time, validity of exponential growth
law, growth yield, metabolic quotient, Effect of substrate concentration, Mo
nod Kinetics,
Determination of K
, Definition of lag period.

B. Estimation of Biomass

Factors influencing choice of method; Dry and wet mass, volume, yields, metabolic rates
light scattering cell count.

C. Stoichiometry

Balance equation, carbon, Nigrogen b
alance, oxidation

reduction principles, product

D. Batch & Plug Flow Culture

Open and closed systems, growth phases, Mathematical model of simple batch culture,
variations in actual practice plug flow culture with and without feedback.

E. Chem
ostat Culture

General principle, Balance equations critical dilution rate, Biomass productivity, comparison
with batch cultures, residence time distribution, Test of validity, imperfect mixing, wall
growth Transient state analysis.

F. Elaboration of Chemos

Turbidostat, pH stat, D.O. stat cultures control mechanisms, Biomass feedback Internal and
External, Chemostat in series Applications.

G. Computer Simulation

Programming with ISIM, writing model equations, Material balance concepts, Kinetic

H. Energy Requirements

Electron transfer concept; maintenance energy, magnitude and control of maintenance
energy, Effect of maintenance.

I. Product Formation in Microbial Cultures

Growth associated and non
growth associated Kinetics, Product
decomposition, Balance
equations for batch and continuous systems, Effect of environmental conditions.

J. Effect of Inhibition and Activation of Growth

Competitive and non
competitive inhibition, Product and substrate inhibition, activators,
Effect on batc
h and continuous systems.

K. Fed
Batch Culture

Analysis, repeated fed
batch culture applications, dialysis culture.

L. Mixed Cultures

Different models and their analysis.

M. Oxygen Demand and Supply

Oxygen demand, solubility, measurement of D.O.T. Redox po
tential oxygen transfer,
measurement of K

N. Aeration and Agitation

Agitation and mixing, Baffled, vortex and airlift systems, Impeller design, Effect of stirring,
sparging and other parameters.

O. Scale up Principles

Methods of scale up and their analy

P. Process Control

Measurement and control of bioprocess parameters like temp., D.O., speed, pH, antifoam
etc., Basic principles of feedback control, Proportional, Integral and derivative control.

MSc Biotechnology



dits: 2

Basic Chemical Engineering calculations. Material balance. Material balance with reactions.
Material balance with recycle and purge. Energy balance. Enthalpy, specific heat, mean
specific heat. Heat Balance. Heat of reaction and heat of solution. M
aterial and Energy
balance together.

Fluid statics.

Fluid mechanics. Potential flow. Newtonian and non Newtonian fluid.

Bingham plastic, pseudo plastic, dilatant.

Time dependent flow

Continuity equation.

Derivation of Bernoulli Equation. Correction for fri
ction losses.

Pump work

Skin friction and fanning friction factor. Laminar flow in pipes.

Hagen Poiseuille Equation. Hydraulic diameter

Flow of incompressible fluid in conduits and thin layers.

Friction loss due to expansion, contraction and fittings.


past immersed bodies. Drag coefficient, terminal velocity. Free and hindered settling.

Metering of fluid flow.

Transportion of fluid. Pumps and valves.

Elementary idea of heat and mass transfer.

MSc Biotechnology



: 4

Molecular Biology

Insertion mutation of a cloned gene

Large/Medium scale isolation of an E. coli plasmid (pUC19 and pUC7 KAPA).

Estimation of amount of plasmid isolated.

Restriction mapping of the plasmid pBR322.

Isolation of a gene (neomycin phosphotr
ansferase) from the plasmid pUC7 KAPA.

Cloning of the BamHI fragment containing the neomycin phosphotransferase gene into the
BamHI site of pUC19 B/W screening.

Transformation of E. coli DH5 ( ) w ith pUC19 and also with pUC19 containing a mutated
line resistance gene.

Isolation of plasmid from the selected transformants by the "miniprep" method.

Restriction analysis of the plasmid to locate position of the insert.

Expression of B
gal under different promoters T7 system and pUC19 (lac promoter), wit
wild type E. coli as control.


Purification of Immunoglobulin from serum

Generation of antibody in mouse

Double diffusion

Conjugation of antibodies with Enzyme

ELISA : i) Capture ELISA

ii) Direct ELISA

Western blot

Affinity column and
purification of antigen

Cell fusion for generation of Hybridoma