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Department of Biotechnology (established in 1980)
presented by
Prof. Ritu Barthwal, Head, Dept. Biotechnology
& Prof. R. P. Singh
COURSES OFFERED
B.Tech. Biotech. 4 years program since 2005
Admission IIT JEE, 200 credit course, Intake 48
M.Sc. Biotech. 2 years program since 1985
Admission JAM, 100 credit courses, Intake 36
Ph.D program since 1981
Admission to GATE/NET qualified, presently 70
FACULTY MEMBERS AND THEIR EXPERTISE
Professor and Head of the Department
Dr. Ritu Barthwal, Biophysics-NMR structure, Molecular Modeling, therapeutic herbs
Professors
Dr. H. S. Dhaliwal, Plant Biotech.-Germplasm Enhancemen, Gene Tagging
Dr. G.S. Randhawa, Genetic Engineering - Molecular Genetics,
Dr. R. P. Singh, Microbial Biotechnology – Microbial Enzymes,
Associate Professors
Dr. R. Prasad, Molecular Biology & Proteomics-Therapeutic Proteins,
Dr. Vikas Pruthi, Molecular Microbiology-Biofilms, Biosurfactants
Dr. Partha Roy, Animal Biotech- Hormone action, Steroid Receptors.
Assistant Professors
Dr. A.K. Sharma, Biochem.-Cloning - expression of plant defense proteins
Dr. Bijan Choudhury, Biochem. Engg - Nitrile Biotransformation, Nutraceuticals
Dr. Pravindra Kumar, Biophysics - Bioinformatics, X-ray Crystallography structures,
Dr. Sanjoy Ghosh, Bioprocess Engg- Biofuel Production, Bioreactor Design
Dr. Naveen K. Navani, Mol. & Chem. Biol.-Aptamers, DNA shuffling.
Dr. Shailly Tomar, Mol. Biol.- Virus Proteins , Structure Analysis.
Dr. Ranjana Pathania, Mol. Microbiology-Antimicrobial Drug Discovery
Dr Maya S. Nair, Biophysics – Biomolecular structure by spectroscopy techniques
M.Sc.students qualifying national – level examination
(no. of students passed every year – 18 to 24)
No. of
students
2003
2004
2005
2006
2007
2008
2009
2010
NET-CSIR/
UGC
12
03
13
12
10
18
16
17
GATE
05
15
18
15
14
10
12
15
DBT-JRF
-
-
04
04
04
-
03
04
PLACEMENT
• Postgraduate and Ph. D. students - R & D companies e.g. Glenmark
Pharma (NJ), Novartis (NJ), Ranbaxy, Panacea Biotech, Dr Reddy’s
Laboratory Hyderabad, Dabur, Cadila, Dr Lal Diagnostic Lab,
Biotech Consortium India Limited, Pepsi foods, Hindustan Lever,
Vam Organics, Wockhardt.
• Most post graduate students qualify GATE/NET/GRE and join Ph.D.
programs in leading universities in India and abroad.
• Several Ph.D. students joined post doctoral program in institutes
abroad such as MD Anderson Cancer Centre Houston (TX), Baylor
College of Medicine Houston (TX), Univ. of California at San
Francisco (UCSF), Univ. of California at San Diego, Museum
National de Histoire Naturelle, Paris (France), John Hopkins Medical
Institute (MD), Harvard Medical School, Univ. of Pennsylvania,
Cleveland University and UIC Chicago.
• On campus recruitment is done centrally for all the disciplines by the
Professor-in-charge Training and Placement.
• B.Tech. students placed at Deloitte, Mu Sigma, T Bits Global, TCS,
3M, Doctoral fellows/Interns at Max Planck Inst Germany, McGill
Univ Canada, Stanford Univ. USA, Toronto Univ. Canada
Post doc/ other visits abroad -17
•
•
•
•
•
•
•
•
•
•
University of Houston, Dept. of Biochemistry
Indo-Russian, ILTP-DST, High performance computing
Cohran Fellowship, US dept. Agriculture
North Dakota state university, NSF project
BOYCAST, DST, Indiana univ. Purdue univ. Indianapolis
BOYCAST, DST, Purdue univ.
Research Astt., Virology, Biol. Sci. Purdue Univ.
Postdoc, Mcmaster univ., Biochemistry & Biomed. Sci.
Postdoc, UMASS medical school, molecular medicine
Postdoc, Imperial college of Science and Technology
2003-2010
•Research publications 150 Intl. J. + 10 Natl. J.
•Research projects 30, Total Rs 450 lakhs
•Patents 5
•Post doc/ other visits abroad 17
•Recognition/ Awards/Honours
•Ph.D awarded – about 15 per year (total
about 150)
0.10
0.08
Absorbance
Berb alone 4.4 M
0.06
Increasing Poly d-(A-T)
concentration
0.04
Berb+Poly d-(A-T) 44M
0.02
*
*
*
0.00
300
450
500
Structure (NMR) – Conformation -Interactions - Biomolecules
Anticancer drugs , Topoisomerase poisons, peptides, flavonoids
Duplex and quadruplex (telomere) DNA
Therapeutic potential of herbs, active principles, characterization
ESI-MS
8000000
Fluorescence Intensity (a.u.)




350
400
Wavelength (nm)
Berb+Poly d-(A-T) 27M
6000000
Increasing Poly d-(A-T)
concentration
4000000
2000000
0
400
Berb alone 4.4 M
450
500
550
600
Wavelength (nm)
650
Palmatine
1H
NMR spectra palmatine- d(CCAATTGG)2
rMD model berberined-(CCAATTGG)2
31P - 31P NOESY
4’-epiadriamycin-d-(CGATCG)2
NOESY Berberine- d-(CCAATTGG)2 complex
NOESY 4’-epiadriamycin
Topoisomerase I inhibition by
aqueous extract of Picrorrhiza kurroa
Restriction inhibition assay of aqueous extract of
Cinnamomum zeylanicum and Picrorrhiza kurroa
with EcoRI restriction endonuclease
Time (min)
Pancreas from (A) streptozotocin-induced diabetic rat
(B) diabetic rat treated with aqueous extract of C. zeylanicum
Blood Glucose (mg/dl)
Aqueous extract
(300 mg/kg body
weight)
Aqueous extract
(200 mg/kg body
weight)
Diabetic control
0
237± 6
303 ± 31*
277 ± 10
60
240 ± 10
305 ± 33*
320 ± 14
120
188 ± 16
299 ± 42*
289 ± 18
180
132 ± 2
240 ± 33*
272 ± 5
240
97 ± 11
209 ± 26*
262 ± 11
300
92 ± 5
162 ± 38*
289 ± 10
% Reduction
61.2
46.5
- 4.3
Short term activity of aqueous extracts of
C. zeylanicum conducted in type 1 diabetic rats
Macromolecular structure by X-ray crystallography,
enzyme kinetics and molecular biology techniques.
# shikimate pathway enzymes # enzymes involved in the
pathway exists in plants and
biodegradation of toxic
micro organisms but absent in
aromatic compounds
humans
Can be a potential target for the
development of antimicrobial
drug for microorganism and
herbicide for plants
 studying enzymes from a
variety of sources including the
different species of bacteria and
plant weeds
•Chemicals having potential to
promote cancer/heart disease &
affect neural development
•Study structure of enzymes
from bacterial pathway that has
partial ability to degrade toxic
aromatic compounds
•Identification of determinants
of substrate specificity & design
improved catalysts for bioremediation, enzyme mechanism
# Chitinases
•Assembly of the fungal cell
wall and human signalling
pathways involved in asthma,
arthritis and cancer
• Reaction mechanism with an
aim to develop potent druglike inhibitors
Biochemical and Structural Characterization of Plant Defense Proteins
# Murraya koenigii Miraculin Like
Protein
# Glycosyl Hydrloase family I
enzyme
# Putranjiva roxburghii Trypsin
Inhibitor.
# Ribosome inactivating proteins
Dnase activity on pBR 322 plasmid
Targeting viral genes of plus sense
ssRNA viruses (Alphaviruses) for
antiviral screening and drug designing
Laboratory Main Gene Targets :
 Capping enzyme
 RNA dependent RNA Polymerase
 Nsp2 replication protease
 Capsid protease
 Protein-Protein interaction: Capsid dimers assemble to form virus particles
 Protein-RNA interaction: Capsid encapsidate viral RNA genome
Laboratory Molecular Tools:
• Molecular gene cloning
• Recombinant protein production
• Function gene characterization
• Development of enzyme assay
• Site directed mutagenesis
• Biophysical and biochemical characterization
• Structure function studies using X-ray crystallography and Bioinformatics
• HTP inhibitor screening
Molecular characterization, functional evaluation and expression of
therapeutic and abiotic stress induce proteins.
• Purification,
molecular characterization of therapeutic ( antimicrobial & antioxidant ) and stress induced proteins.
•Cloning , characterization and over
expression of candidate genes using
RNAi technology.
• Functional evaluation of the respective
proteins using various assays.
SDS-PAGE & 2-D Protein Profile
Immuno-localization study
of 31.6 kDa protein
Antimicrobial activity assay of
protein
Microorganisms
Diameter of Inhibition Zone (mm)
Amount of protein per well
(μg)
Standard
20
Kan (30μg/well)
40
60
80
Bacteria
Serratia macescens
(MTCC2453)
12
14
17
20
26
Pseudomonas putida
(MTCC2453)
12
15
18
21
28
Staphylococcus aureus
(MTCC2940)
13
16
18
21
30
Bacillus subtilis
(MTCC 2423
12
14
17
19
26
Fungi
AmB(30μg/well)
Aspergillus niger
(ITCC 5454)
10
12
13
15
23
Candida Albican
(MTCC 227)
10
11
13
14
23
Identification of 31.6 kDa protein by mass spectrometry
Regeneration of pancreatic cells by guggulsterone
Isolated mesenchymal stem cells from rat bone marro
Molecular Endocrinology and Reproductive Physiology- cell based assays
to screen compounds, understand, both in vitro (cell lines) and in vivo (rodent models), mode of
action.Endocrine disruptors: environmental chemicals (pesticides, industrial chemicals, various
chemicals,mainly steroids).;Plant based medicines: bioactive principles from medicinal plants
for diseases like diabetes, cancer and infertility.;Stem cells and their differentiation: Isolation
of mesenchymal stem cells, role of various hormones and their pathways for the differentiation
of adipocytes and osteoblasts.
Pterostilbene (isolated
from Pterocarpus
marsupium) as
anticancer agent
Toxic effects of
Triclosan (a potent
Endocrine disruptor)
on sperm structures
Control
Control
Treated
Treated
MICROBIAL PRODUCTION OF ENZYMES, ENZYME
ENGINEERING AND APPLICATIONS
a
b
c
121 k Da
77 k Da
40 k Da
29 k Da
SEM of P. oxa & Plu. ostr.
SDS-PAGE (a), ZYMO; XYL (b) LACC (c)
1st D
2nd D
3rd D
4th D
5th D
COMPATIBILITY ASSAY
MAJOR ACTIVITIES:
1300
a
Xylanase
Laccase
Xylanase (IU ml-1)
1100
30
1000
900
25
800
700
-1
Laccase (IU ml )
1200
35
b
20
•Exploration of microbial diversity for the
production of enzymes of industrial
significance
•Biopolymers, targeted nano drug delivery
system, anti microbial reagents
600
500
15
Axenic
culture
Co-culture
IMPROVED XYL AND LACC PRODN. USING
CO-CULTIVATION
FTIR ANALYSIS (a) UNTREATED
(b) TREATED
•Engineered
microbial
strains
and
development of microbial consortia for
bioremediation of defined
industrial
effluents
MICROBIAL PRODUCTION OF SURFACE ACTIVE AGENTS
USING CHEAP RAW MATERIAL
CHEAP RAW MATERIALS
Cotton seed
SCREENING METHODS
Bamboo Powder
BIOSURFACTANT PRODUCERS
Baggase powder Acacia powder
APPLICATIONS
Blood Agar
Drop collapse
Tensiometer
Emulsification
B. subtilis, P. aeruginosa, Lactobacillus spp,
Candida spp, A. calcoaceticus etc.
CHARACTERIZATION
Emulsifier, Demusifier, Solublizer
Thickeners, Wetting and Foaming agent
ENHANCED BIOSURFACTANT
PRODUCTION
COSMETICS
Health care &
Adhesives &
Beauty products
antiperspirants
Soaps,
toothpastes
shampoo,
conditioners
SEM
GC-MS
FTIR
(
A
)(
B
)
Scale up process
NMR
Contact: Dr. Vikas Pruthi
[email protected]
Molecular Analysis of Candida Biofilm
Contact: Dr. Vikas Pruthi
[email protected]
Microscopic analysis
(CLSM, SEM, AFM)
Candida Biofilm
Drug efflux
pump studies
EPS Purification
(GC-MS, HPLC,
Ion exchange)
Biomaterial
interaction
EPS analysis
(Carbohydrate,
protein,
phosphorus)
Immunological
studies
Biofilm Quantification
(XTT, Dry weight,CFU)
Gene analysis
Effect of Surfactant,
Herbal preparations,
Enzymes, Antifungals
Structural analysis
(IR, NMR)
Enzymatic surface modification of Polyacrylonitrile
Enzymatic surface modification of
Polyacrylonitrile using nitrile metabolizing enzymes of Amycolatopsis sp.
Advantages of using enzymatic surface
modification:
Greener route
Chemo Specificity
Increase in hydrophilicity of polymer
Ambient reaction conditions
Negligible effect on polymer strength
properties
FTIR spectra of enzyme treated Polyacrylonitrile and control
Fermentative Production of trehalose using P. shermanii NCIM5137 in different carbon sources
500
Trehalose, a low calorie sugar having
nutraceutical properties was attempted to
produce by fermentation using P. shermanii
NCIM 5137.
Glycerol from bio-diesel waste found to improve
trehalose yields based on biomass (Yt/x) and substrate
consumed (Yt/s).
400
300
200
100
0
glucose sucrose glycerol
b iodies
el waste
Yt/x(mg/gm)
87
171
381
404
Yt/s(mg/gm)
1.3
5.1
2.8
104
Maximum trehalose production in various carbon sources
•Development of detection kits for microbial pathogens using
nucleic acid aptamers.
In vivo combinatorial selection of microbial strains for detection of
pollutants using promoter engineering.
Selection of lactic acid bacteria with enhanced probiotic attributes.
Aptamer generation against Salmonella typhi -FACS Confirmation
Random DNA
+
Salmonella typhi
ST1 aptamer DNA
+
Salmonella typhi
ST1 aptamer DNA
+
Escherichia coli
In-vivo SELEX Based Discovery of Synthetic Promoters for Biosensing
Sig E 98 (sigE)
NNNNNBVAACHMNNAAAAANNNNNNNNNTCNNAHHWWMMNNNNN
B=GCT, H=ACT W=AT Y=CT M=AC
Fig. 1 :Multiple sequence alignment of stress responsive promoters
Fig.2: Library Construction
Library
cloned
rRygC as Reporter system
Least background
+ve selection of Inducible
Promoter
Fig . 4 : Construction of Toxic Peptide based Promoter Probe vector
Fig.3 RygC- rRygC Toxin Antitoxin system
2
A pNYL-rygc
without
promoter
1
uninduced
10
8
6
4
0
2
Fig.5 : Clone Confirmation
1,3,5,7,9,11– Cut clone plasmid
2,4,6,810 – Uncut clone plasmid
Response of NAL promoter at different
concentration of Nalidixic acid
O.D. at A600 nm
1 2 3 4 5 6 7 8 9 10 11 12
B pNYL-rygc
with NAL
responsive
promoter
Concentration of Nalidixic acid µg/ml
1-pNYL-Rygc (without Promoter ) Induced by 6ug/ml
Nalidixic Acid 2-pNYL-Rygc(without Promoter )
Uninduced 3-pNYL-NA( Nalidixic Acid responsive
Promoter) Induced by 6ug/mlNalidixic Acid
4-pNYL-NA (Nalidixic Acid responsive Promoter)
uninduced
Fig.6: Nalidixic acid responsive Promoter discovered through In-Vivo SELEX. Figure depicts Live and Dead
21 selection
achieved for this synthetic promoter while screening the entire promoter library with sublethal concentration of
Genotoxic agents.
A Microbe Isolated from Uttarakhand’s Soil
Degrades Multiple Pesticides
1
1
A
Fig 1. Light Microscopy at 100X
Fig 2. Scanning Electron
microscopy at 20000X
2
3
B
3
2
Fig 3. 1)Pseudomonas aeruginosa 2) E. coli (negative control) 3) B.
subtilis(negative control) A) Minimal media and imidacloprid as sole
carbon B) Minimal media and endosulfan as sole carbon source
A
A
Endosulfan
Imidacloprid
B
Nitrosourea
Imidaclopri
d
B
Endosulfan
Endosulfan
ether
Fig 4. Growth Profiles of RPT-52 in
minimal media supplemented with 0.5
mM pesticide (▬ Imidacloprid, ■Endosulfan)
Fig 5. ESI-MS Spectra of A) Minimal
Media and Imidacloprid B) Minimal
Media and Imidacloprid in the presence
of pesticide degrading bacteria
Fig 6. ESI-MS Spectra of A) Minimal
Media and Endosulfan B) Minimal Media
and Endosulfanin the presence of
pesticide degrading bacteria
Identification and Characterization of Small RNA
in Acinetobacter baumannii
1000bp
800bp
600bp
31 Predictions
1 2 3
1 2 3
1000bp
800bp
600bp
400bp
300bp
400bp
300bp
200bp
200bp
100bp
100bp
5S rRNA
Fig 3A.
*AbsR 25
small RNA
Fig 1. Gram
Stained cells under
100X magnification
Fig 2. Growth curve of
Acinetobacter baumannii.
Fig 3B.
AbsR 28
small RNA
Lane 1- 1.6 OD Total RNA (20ug)
Lane 2- 1.0 OD Total RNA (20ug)
Lane 3- 0.4 OD Total RNA (20ug)
*Acinetobacter baumannii Small RNA
(a)
(I) Bioethanol production
12
7
Biothanol production, Dry cell biomass
production and Total reducing sugars
consumption (g/l)
100
90
80
70
60
50
40
30
20
10
0
5
4
3
2
1
dry cell Biomass (g/l)
6
144
136
128
120
112
96
104
88
80
72
64
56
48
40
32
28
24
0
0
Conc. of reducing sugars
and ethanol (g/l)
Overview:
Substrate: Lignocellulosic perennial grass
Fermentation condition: pH: 5, Temp.: 30°C, rpm:
200
Microorganisms: P.stipitis, T.reesei, S.cerevisiae
Tim e in hours
(b)
Total reducing sugars (g/l)
Ethanol (g/l)
(II) Microbial production
of Phytase
Dry cell biomass
(c)
10
8
6
4
2
0
0
2
4
6
8
Dry cell Biomass (g/l)
12 16 20 24 28 32 36 40
Tim e in hours
Ethanol (g/l)
44 48 52 56 64
Total reducing sugars (g/l)
RESEARCH FOCUS
• Structural Biology and Drug Discovery: Structure-based drug
designing (NMR, x ray crystallography & molecular
modeling)- anticancer agents acting on DNA, proteins /DNA /
RNA as drug targets, herbs having therapeutic potential,
protein-protein and protein-nucleic acid interactions,
metalloenzymes, phytases, esterases, trypsin inhibitors,
antimicrobial peptides, characterization of proteins and gene
cloning, high throughput screening for inhibitors for using
genomic library, aptamer technology for drug target
identification, enzymes of biosynthetic pathway of pathogens,
proteins with biotic and abiotic resistance, salt tolerance, cell
based assays, steroid receptors, effects of endocrine disrupting
chemicals, cell assays to screen the compound, screening
insulin receptors sensitizer, stem cell- biomaterial interactions.
• Microbial Technology, Bioprocess technology and
Nanobiotechnology; enzyme production and engineering,
biobleaching, designing of strain engineering for
bioremediation, biofilms, biosurfactants, promoter engineering
for biosensors, biopolymers, designing of nanoparticles and
drug targeting, small RNA and gene regulation, biofuels,
nitrile bioremediation
MAJOR EQUIPMENT / FACILITIES
HPLC, Protein Purification System, uv-vis spectrophotometer,
spectrofluorimeter, Gas Chromatograph
Fermentor, Bioreactors, Orbital Shaker& Incubator Shaker, Sonicator
DNA Synthesizer & Peptide Synthesizer
Electrophoresis System, Transilluminator with Polaroid Camera, Gel
Documentation & Analysis
Silicon Graphics workstations, Software INSIGHT II, DISCOVER, AMBER,
MOE, FELIX, CURVES, SPHINX-LINSHA, CNS-XPLOR, HYPERCHEM .
Thermal Cycler (PCR), Elisa Plate Reader
Plant Growth Chamber & CO2 Incubator, Ultracentrifuges
Inverted, Stereozoom and Fluorescence Microscope
500 MHz FT NMR, cryoprobe & LC-NMR-MS(ESI MS), Software TOPSPIN
1.3, NMR REFINE PLUS - DGII & NMR XPLOR , FELIX, INSIGHT II ,
DISCOVER, BIOPOLYMER & GAUSSIAN
Single Crystal X-Ray Diffractometer with CCD Detector Model
Fluorescence Lifetime,TEM, SEM, EPMA, ICP-MS, TIMS, DTA, etc.