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 2005Admission IIT JEE, 200 credit course, Intake 48

M.Sc. Biotech. 2 years program since 1985Admission 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 DepartmentDr. Ritu Barthwal, Biophysics-NMR structure, Molecular Modeling, therapeutic herbs

ProfessorsDr. 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 ProfessorsDr. R. Prasad, Molecular Biology & Proteomics-Therapeutic Proteins, Dr. Vikas Pruthi, Molecular Microbiology-Biofilms, Biosurfactants Dr. Partha Roy, Animal Biotech- Hormone action, Steroid Receptors.

Assistant ProfessorsDr. A.K. Sharma, Biochem.-Cloning - expression of plant defense proteinsDr. Bijan Choudhury, Biochem. Engg - Nitrile Biotransformation, Nutraceuticals Dr. Pravindra Kumar, Biophysics - Bioinformatics, X-ray Crystallography structures, Dr. Sanjoy Ghosh, Bioprocess Engg- Biofuel Production, Bioreactor DesignDr. 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 DiscoveryDr 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

•Post doc/ other visits abroad 17

•Research publications 150 Intl. J. + 10 Natl. J.

•Research projects 30, Total Rs 450 lakhs

•Recognition/ Awards/Honours

•Ph.D awarded – about 15 per year (total about 150)

•Patents 5

300 350 400 450 500

0.00

0.02

0.04

0.06

0.08

0.10

Berb+Poly d-(A-T) 44μM

* **

Increasing Poly d-(A-T) concentration

Berb alone 4.4 μM

Abso

rban

ce

Wavelength (nm)

400 450 500 550 600 6500

2000000

4000000

6000000

8000000Berb+Poly d-(A-T) 27μM

Berb alone 4.4 μM

Increasing Poly d-(A-T) concentration

Flu

ores

cenc

e In

tens

ity (a

.u.)

Wavelength (nm)

Structure (NMR) – Conformation -Interactions - BiomoleculesAnticancer drugs , Topoisomerase poisons, peptides, flavonoidsDuplex and quadruplex (telomere) DNATherapeutic potential of herbs, active principles, characterization

ESI-MS

NOESY Berberine- d-(CCAATTGG)2 complex

1H NMR spectra palmatine- d(CCAATTGG)2

rMD model berberine-d-(CCAATTGG)2

31P - 31P NOESY 4’-epiadriamycin-d-(CGATCG)2

NOESY 4’-epiadriamycin

Palmatine

Topoisomerase I inhibition by aqueous extract of Picrorrhiza kurroa

Time (min) 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

Restriction inhibition assay of aqueous extract of Cinnamomum zeylanicum and Picrorrhiza kurroa

with EcoRI restriction endonuclease

Pancreas from (A) streptozotocin-induced diabetic rat(B) diabetic rat treated with aqueous extract of C. zeylanicum

Short term activity of aqueous extracts of C. zeylanicum conducted in type 1 diabetic rats

# shikimate pathway enzymespathway exists in plants and

micro organisms but absent in humansCan 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

# 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 drug-like inhibitors

Macromolecular structure by X-ray crystallography, enzyme kinetics and molecular biology techniques.

# enzymes involved in the biodegradation of toxic aromatic compounds•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 biorem-ediation, enzyme mechanism

Biochemical and Structural Characterization of Plant Defense Proteins

# Murraya koenigii Miraculin Like Protein # Putranjiva roxburghii Trypsin

Inhibitor.

# Glycosyl Hydrloase family I enzyme

# 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 enzymeRNA dependent RNA PolymeraseNsp2 replication proteaseCapsid proteaseProtein-Protein interaction: Capsid dimers assemble to form virus particlesProtein-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

SDS-PAGE & 2-D Protein Profile

Antimicrobial activity assay of protein

Microorganisms Diameter of Inhibition Zone (mm)

Amount of protein per well (μg)

Standard

20 40 60 80 Kan (30μg/well)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

Immuno-localization study of 31.6 kDa protein

Identification of 31.6 kDa protein by mass spectrometry

Molecular characterization, functional evaluation and expression of therapeutic and abiotic stress induce proteins.

• Purification, molecular characterizati-on of therapeutic ( antimicrobial & anti-oxidant ) and stress induced proteins.•Cloning , characterization and over expression of candidate genes usingRNAi technology.• Functional evaluation of the respectiveproteins using various assays.

Regeneration of pancreatic cells by guggulsterone 

Pterostilbene (isolated from Pterocarpus marsupium) as anticancer agent 

Isolated mesenchymal stem cells from rat bone marro

Control Control

TreatedTreated

Toxic effects of Triclosan (a potentEndocrine disruptor)on sperm structures 

Molecular Endocrinology and Reproductive Physiology- cell based assaysto screen compounds, understand, both in vitro (cell lines) and in vivo (rodent models), mode ofaction.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: Isolationof mesenchymal stem cells, role of various hormones and their pathways for the differentiationof adipocytes and osteoblasts.

SEM of P. oxa & Plu. ostr.

MICROBIAL PRODUCTION OF ENZYMES, ENZYME ENGINEERING AND APPLICATIONS

1st D 2nd D 3rd D 4th D 5th D

COMPATIBILITY ASSAY

Xyl

anas

e (I

U m

l-1)

500

600

700

800

900

1000

1100

1200

1300

Lac

case

(IU

ml-1

)

15

20

25

30

35

XylanaseLaccase

Axenic culture

Co-culture

IMPROVED XYL AND LACC PRODN. USING CO-CULTIVATION

121 k Da

77 k Da

40 k Da

29 k Da

FTIR ANALYSIS (a) UNTREATED (b) TREATED

a

b

SDS-PAGE (a), ZYMO; XYL (b) LACC (c)

ba c

•Exploration of microbial diversity for the production of enzymes of industrial significance

•Biopolymers, targeted nano drug delivery system, anti microbial reagents

•Engineered microbial strains anddevelopment of microbial consortia forbioremediation of defined industrialeffluents

MAJOR ACTIVITIES:

BIOSURFACTANT PRODUCERS

CHEAP RAW MATERIALS

(A)(B)

CHARACTERIZATION

SEM FTIR

GC-MS NMR

B. subtilis, P. aeruginosa, Lactobacillus spp, Candida spp, A. calcoaceticus etc.

SCREENING METHODS

Blood Agar Drop collapse

Tensiometer Emulsification

ENHANCED BIOSURFACTANT PRODUCTION

Cotton seed Bamboo Powder

Baggase powder Acacia powder

Scale up process

APPLICATIONS

Emulsifier, Demusifier, Solublizer Thickeners, Wetting and Foaming agent

COSMETICS

Health care & Beauty products

Soaps, toothpastes shampoo, conditioners

Adhesives & antiperspirants

MICROBIAL PRODUCTION OF SURFACE ACTIVE AGENTS USING CHEAP RAW MATERIAL

Contact: Dr. Vikas Pruthi vikasfbs@iitr.ernet.in

Molecular Analysis of Candida Biofilm

Microscopic analysis(CLSM, SEM, AFM)

Candida Biofilm

Biomaterial interaction

Biofilm Quantification(XTT, Dry weight,CFU)

Structural analysis(IR, NMR)

Drug efflux pump studies

EPS Purification(GC-MS, HPLC, Ion exchange)

Immunologicalstudies

Gene analysis

Effect of Surfactant, Herbal preparations,

Enzymes, Antifungals

EPS analysis(Carbohydrate,

protein, phosphorus)

Contact: Dr. Vikas Pruthi

vikasfbs@iitr.ernet.in

Enzymatic surface modification of Polyacrylonitrile using nitrile metabo-lizing enzymes of Amycolatopsis sp.Advantages of using enzymatic surfacemodification:

Greener routeChemo Specificity Increase in hydrophilicity of polymerAmbient reaction conditionsNegligible effect on polymer strength

properties FTIR spectra of enzyme treated Polyacrylonitrile and control

Enzymatic surface modification of Polyacrylonitrile

Fermentative Production of trehalose using P. shermanii NCIM5137 in different carbon sources

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 substrateconsumed (Yt/s).

Maximum trehalose production in various carbon sources

0

100

200

300

400

500

Yt/x(mg/gm) 87 171 381 404

Yt/s(mg/gm) 1.3 5.1 2.8 104

glucose sucrose glycerolb iodiesel waste

•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

21

Sig E 98 (sigE)NNNNNBVAACHMNNAAAAANNNNNNNNNTCNNAHHWWMMNNNNNB=GCT, H=ACT W=AT Y=CT M=AC

1 2 3 4 5 6 7 8 9 10 11 12

0

1

2

2 4 6 8 10un

indu

ced

O.D

. at A

600

nm

Concentration of Nalidixic acid µg/ml

Response of NAL promoter at different concentration of Nalidixic acid

A pNYL-rygc without promoter

B pNYL-rygc with NAL responsive promoter

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

rRygC as Reporter systemLeast background+ve selection of Inducible Promoter

Fig.5 : Clone Confirmation1,3,5,7,9,11– Cut clone plasmid2,4,6,810 – Uncut clone plasmid

In-vivo SELEX Based Discovery of Synthetic Promoters for Biosensing

Library cloned

Fig. 1 :Multiple sequence alignment of stress responsive promoters

Fig . 4 : Construction of Toxic Peptide based Promoter Probe vector

Fig.2: Library Construction

Fig.3 RygC- rRygC Toxin Antitoxin system

Fig.6: Nalidixic acid responsive Promoter discovered through In-Vivo SELEX. Figure depicts Live and Dead 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

Fig 1. Light Microscopy at 100X Fig 2. Scanning Electron microscopy at 20000X

1

2 3A

1

2 3B

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

Fig 4. Growth Profiles of RPT-52 in minimal media supplemented with 0.5 mM pesticide (▬ Imidacloprid, ■-Endosulfan)

NitrosoureaImidacloprid

B

Imidacloprid

A

Endosulfan ether

EndosulfanA

B

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

Endosulfan

Identification and Characterization of Small RNA in Acinetobacter baumannii

1    2    3 1    2    3

Fig 3A. *AbsR 25 small RNA

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

31 Predictions

5S rRNA

1000bp800bp600bp

400bp300bp

200bp

100bp

1000bp800bp600bp

400bp300bp

200bp

100bp

Fig 1. Gram Stained cells under 100X magnification

Fig 2. Growth curve of Acinetobacter baumannii.

(I) Bioethanol productionOverview:Substrate: Lignocellulosic perennial grassFermentation condition: pH: 5, Temp.: 30°C, rpm: 200Microorganisms: P.stipitis, T.reesei, S.cerevisiae

(b) (c)

(a)

(II) Microbial production of Phytase

RESEARCH FOCUS• Structural Biology and Drug Discovery: Structure-based drug

designing (NMR, x ray crystallography & molecularmodeling)- anticancer agents acting on DNA, proteins /DNA /RNA as drug targets, herbs having therapeuticpotential, protein-protein and protein-nucleic acidinteractions, metalloenzymes, phytases, esterases, trypsininhibitors, antimicrobial peptides, characterization of proteinsand gene cloning, high throughput screening for inhibitors forusing genomic library, aptamer technology for drug targetidentification, enzymes of biosynthetic pathway ofpathogens, proteins with biotic and abiotic resistance, salttolerance, cell based assays, steroid receptors, effects ofendocrine disrupting chemicals, cell assays to screen thecompound, screening insulin receptors sensitizer, stem cell-biomaterial interactions.

• Microbial Technology, Bioprocess technology andNanobiotechnology; enzyme production andengineering, biobleaching, designing of strain engineering forbioremediation, biofilms, biosurfactants, promoter engineeringfor biosensors, biopolymers, designing of nanoparticles anddrug targeting, small RNA and gene

MAJOR EQUIPMENT / FACILITIES

HPLC, Protein Purification System, uv-vis spectrophotometer, spectrofluorimeter, Gas ChromatographFermentor, Bioreactors, Orbital Shaker& Incubator Shaker, SonicatorDNA Synthesizer & Peptide SynthesizerElectrophoresis 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 & GAUSSIANSingle Crystal X-Ray Diffractometer with CCD Detector ModelFluorescence Lifetime,TEM, SEM, EPMA, ICP-MS, TIMS, DTA, etc.