TDC TRAINING IISC, KUDAPURA CAMPUS,

CHALLAKERE, KARNATAKA, INDIA

CoordinatorProf. M.S. Hegde

Biology CoordinatorsProf. S.Shishupala, HOD, Dept of Microbiology,

Davangere University, Davangere &

Dr. Aravinda, Crystallographer, IISC, Bangalore

TraineeDr. Mousumi DasAsst Professor,

Dept of Biotechnology, SIT, Tumkur

WHY TDC, IISC Challakerere ?

• Indian Institute of Science - Challakere Campus at Kudapura

• Indian Institute of Science Bangalore celebrated its centenary in the year

2009. To expand its activities, need for a second campus was felt at that

time.

• Indian Institute of Science has acquired a second campus at Kudapura,

Challakere, Chitradurga district of Karnataka.

• The creation of the second campus has been greatly facilitated by the

Government of Karnataka.

• The campus is located at Kudapura 15 Km from Challakere on the

Challakere - Nayakanahatti Road. Kudapura campus is 220 Km away from

the Bangalore campus.

Talent Development Centre (TDC)The Institute conceived the idea of starting a Talent Development Centre mainly to impart training to science teachers at all levels, to conduct discussion meetings, seminars, winter and summer schools, provide academic and research facilities for the young high school, college, and University students. High School Science Teachers Training Program is considered a priority because it is in the high schools the students are introduced to science. Science education is crucial for the development of our Nation. It is the experience of the Institute that if the teachers are trained, vast number of students gets benefitted for a long period of time. 

Vision of High School teachers TrainingThe primary vision of this program is teaching and training teachers to teach students with passion. Motivating them to have higher level of intellectual activity is more important than just giving lectures to the participating teachers and covering the course syllabus. The hallmark of the IISc academic program is in its rigor, honesty of purpose, simplicity, humility, sincerity, time consciousness, hard work and delivery. It is the research and the research attitude that drives the Institute. If an event is announced in IISc it takes place at the scheduled time. Can we inculcate IISc's culture of learning in these teachers ? 

Non Teaching Staffs , TDC, IISC, Kudapura Campus,Challakere

IISC , TDC , KudapuraOutside and InsideClassrooms and Well Facilitated Lab with all necessaru basic amenities

Biology and Chemistry Lab

Entrance

IISC , TDC , Kudapura campus ;Outside view

Biology and Chemistry Lab

Biology Lab Biology Lab with a trainee

TDC Entrance

Theory lectures scheduled from 23rd Nov to 13th Dec.,2016

Practicals scheduled from 23rd Nov to 13th Dec.,2016 represented in a Lab Manual

List of Practicals Carried out from 23rd Nov to 13th Dec,2016

Topics covered in theory lectures by resource persons

Topics covered in theory lectures by resource persons

Topics covered in theory lectures by resource persons

List of experiments covered in practicals

List of experiments covered in practicals

Dr. S. Shishupala, HOD, Dept of Microbiology, Davangede Univ. Davangede; Biology Coordinator at TDC

Prof. Dipak Saini , Dept of Biological Sciences, IISC, Bangalore

Prof. D.N Rao, Dept of BiochemistryIISC, Bangalore

Prof. P.Kondaiah, Dept of Reproductive Biology and Gene Expression Unit

IISC, Bangalore

Prof. P.Kondaiah, Cancer BiologistIISC, Bangalore

Prof. M.R.N. Murthy, IISC, Bangalore

Some of the Resource Persons… In camera

Prof. Upendra Nongthomba, DBGL Lab, IISC, Bangalore

Prof. R .Manjunath, Associate Professor, Dept of Biochemistry, IISC Bangalore

Associate ProfessorJNCASR ,Jakkur, Bangalore 560 064, India

Dr. Hemalatha Balaram

Persons behind the hardships of training being conducted in TDC,IISC,Kudapura, Challakere

Brief Glimpses of TDC , IISC, Kudapura, Challakere from 23rd Nov to 13th Dec,2016

ESTABLISHMENT OF LAB FOR BIOTECHNOLOGY WITH BASIC AND HIGHEND INFRASTUCTURE WITH A

NOMINAL COSTING

Instruments Approximate Costing(Rupees)

Glasswares Miscellaneous ( Rupees) Funds and Collaborative agencies suggested

Total costing

Microscopes and Autoclave and pressure cooker

50,000 Conical flasks -50 ml to 1000 ml (Borosil)

Micro Pippettes (0.0 to 5 ml) Tarsons - 5,000

CSR –corporate Social Research funds from near industries

4.0 -6.0 lakhs

Hot Air Oven 8,000 Beakers (10-1000 ml; Borosil),

Pippette stand, Cuvette stand, Testtube stand ,Eppendorfs tubes (1 ml to 5 ml) and stands, micropippete tips and (0-5 ml),rubber bands, dialysis bag,forceps, needles, spatula, centrifuge tubes, , etc

India Bioscience KSITBTVGST

Plastic beakers (500 to 1000 ml) - Tarsons

Shaker Incubator/ Vortex mixers

10,000 Glass pippettes (1 ml to 10 ml)-Borosil

Disposable autoclavable bags,

LAF/pH meter (digital) 48,000 Funnels (small, medium and big size) – glass and plastic made

micropippete tip boxes

Cooling Centrifuge/ Table top centrifuge and microcentrifuge

65,000 BOD bottles (100 ml – 1000 ml)

gel staining and destaining box,

Dessicators for chemical storage

8,000 Storage bottles Blubber for acid pippeting

PCR or Thermocycler and Powerpack combined PAGE unit

30,000-40,000

Volumetric flask Columns for chromatography

Spectrophotometer/ Colorimeter

47,000 Separating funnel Blotting papers, Whattmann Filetre paper, pH strips (core and decibel range 1.0 to 14.0)

DNA gel running machine or Gelrocker and illuminator

Glass slides, cavity slides, coverslips etc

Total- 1.0to 4.5 lakhs

Total-1.5 lakhs Total-– 30,000 to 50,000

Some lab amenities starting from very basic to highend

Fluorescence Microscope StereoBinocular

Microscope

Sterilization efficiency checking materials for autoclave

Filter sterilization and bacteriophage filter elements

Eppendorf tube stand to keep in waterbath

SDS PAGE UNIT

Shaker Incubator

Gas Chromatographer

Shaker Waterbath

Arrangements and designing of feasible accomodation of chemicals and glasswares with a brief systematic planning

Infrastucture facilities with maximum utilization in minimum space

Glimpses of theory lectures on some basic theoretical issues – common perspective of Lifesciences including all branches

Mass is the amount of matter in an object.Difference Between Mass and WeightStudents of physics often confuse mass and weight of an object and many also feel that there is no difference between the two, while the fact is that there is a lot of difference between the two.Mass is the amount of matter present in a body and is an intrinsic property of the body. Mass of an object remains the same always at any place.Weight on the other hand is the force which a given mass feels due to the gravity at its place. Weight is measured in units of Force like Newton (which is the SI unit of Force).If your mass is 60 kgs then your weight is approximately 60 x 10 = 600 Newtons. This is becauseForce = mass x acceleration (From Newton's second Law)Thus, weight = mass x acceleration due to gravityIf you go to moon your mass remains same, i.e 60 kgs, but your weight becomes less by 1/6 amount, since moon's gravity is 1/6 that of earth.Mass of a body is measured by balancing it equally with another known amount of mass. You keep known amount of masses like blocks of 1 kg, 2 kg etc on one side till both the sides balance and then add up the numbers on the known side of mass and thus calculate the unknown mass. This works because, when the masses are equal on both the sides of the balance the effect of gravity cancels out for both (i.e weight cancels out) and hence we can calculate the mass on one side of the balance if we know the mass on the other side of the balance.Weight is measured using a scale which effectively measures the pull on the mass exerted by the gravity of the earth.

Ion type Composition M/z ratio Frequency

A Σ +H –CO S-27 quite common

B Σ +H S+1 common

C Σ +H +NH +H +H S+18 rare

X Σ +OH +CO S+45 rare

Y Σ +OH +H +H S+19 very common

Z Σ +OH –NH very rare

doubly-charged parent parent ion +H+ (parent M/z + 1) / 2 very common

trebly-charged parent parent ion +H+ +H+ (parent M/z + 2) / 3 rare

internal ion Σ +OH +H +H S+19 rare

immonium ion Σ +H -CO S-27 rare

Table 1. Masses of ions found in tandem spectra.'Σ' here denotes the total mass of the constituent amino acids, as given in table 2.The Frequency column applies to low-energy collisions in a modern QTOFspectrometer, higher-energy collisions in older spectrometers gave rise to agreater variety of ions.

S+2

letter name mass,DaG glycine 57.02A alanine 71.04S serine 87.03P proline 97.05V valine 99.07T threonine 101.05C cysteine 103.01I isoleucine 113.08L leucine 113.08N asparagine 114.04D aspartic acid 115.03Q glutamine 128.06K lysine 128.09E glutamic acid 129.04M methionine 131.04H histidine 137.06F phenylalanine 147.07R arginine 156.10Y tyrosine 163.06W tryptophan 186.08

carboxymethyl cysteine 161.05carbamidated cysteine 160.03oxidised methionine 147.04

Table 2. Masses of amino acids to two decimal places.

Parent ion mass 1275.67

Parent++

Step 1. Identify doubly charged parent ion(1275.67 + 1)/2 = 638.33

We do this to just eliminate this peak and not associate it with a fragment ion. This has no other purpose

Parent++

Yn-1

Y

•Step 2. Identify high mass peaks at right end of spectrum•These could be Y ions•1112.70 – could be Yn-1 arising from cleavage of amino terminal amino acid from parent ion Yn•Parent ion is +19 (18+1(singly charged))•1275.67-1112.70 = 162.97•162.97 could correspond to a single amino acid or the sum of masses of a small number of amino acids.•162.97 = mass of tyrosine

Parent++

Yn-1

YG

Yn-2

Yn-3

Yn-4

Yn-5

I/L

V

T

•Identify Yn-2 and Yn-3•1112.70-1055.67 = 57.03 G•1055.67 – 1014.52 = 41.15 not consistent with any amino acid, peak small, we skip it.•1055.67 – 947.67 = 113.05 I/L•Similarly the next two are valine and threonine•942.62-843.48 = 99.14 Valine•843.48 – 742.46 = 101.02 threonine

Ion Sequence predicted mass

A1 Y 136.08

A2 YG 193.10

A3 YGI 306.18

A4 YGIV 405.25

A5 YGIVT 506.30

B2 YG 221.09

B3 YGI 334.18

B4 YGIV 433.25

B5 YGIVT 534.29Table 3.

B1 cannot form

Parent++

Yn-1

YG

Yn-2

Yn-3

Yn-4

Yn-5

I/L

V

TA1

A5

A3A2

A4

B2

B3

B4

B5

Parent++

Yn-1

YG

Yn-2

Yn-3

Yn-4

Yn-5

I/L

V

TA1

A5

A3A2

A4

B2

B3

B4

B5

Parent++

Yn-1

YG

Yn-2

Yn-3

Yn-4

Yn-5

I/L

V

TA1

A5

A3A2

A4

B2

B3

B4

B5

Y0 Y0Y0

•Peaks at 843, 942, 1055 all have peaks 18 Da to the left of them•Due to loss of water•These are designated as Y0

•Ser or Thr following(?) YGIVT

Parent++

Yn-1

YG

Yn-2

Yn-3

Yn-4

Yn-5

I/L

V

TA1

A5

A3A2

A4

B2

B3

B4

B5

Y0 Y0Y0

++Parent0

++Parent*

•Small peaks at 629.33 and 629.87•Other doubly charged ions Parent0/parent’•Drop of water (-18) (S or T)•Drop of ammonia (-17) (Q, N, K, R)•Tryptic peptides contain K/R

Chemistry for Biology

Problems and solutions derivations on pH, pka, % dissociation, molarity, molality, preparation of buffers ; evaluation of kw etc.

HOW DOCK WORKS

Step 1: Start with crystal coordinates of target receptor

In this example, HIV-1 protease is the target receptor, with its active site aspartyl

groups identified in red.

Step 2: Generate molecular surface for receptorThis is performed using Mike Connolly's ms program. Note that only the surface for the active site needs to be generated

For the rest of this overview, we'll use a a blow up of the active site

Step 3: Generate spheres to fill the active site

The shape of cavities in the receptor is used to define spheres; the centers of the spheres become potential localtions for ligand atoms.

In the following picture, the sphere centers are identified by cyan triangles, and the sphere surfaces are shown:

Side view of spheres

Step 5: Scoring

Each oriented molecule is then scored for fit. There are currently 3 scoring schemes:

·Shape scoring -- which uses a loose approximation to the Lennard-Jones potential

·Electrostatic scoring -- which uses the program DELPHI to calculate electrostatic potential ·Force-field scoring, which uses the AMBER potential

In this example, this is the top-scoring orientation for the molecule thioketal in the HIV1-protease active site, using force-field scoring. (please note, this is a different orientation than previously published, as those runs were done using the shape scoring scheme, and a different version of the protease crystal structure

Final notes Here is a comparison of the top scoring orientation of the molecule thioketal with the orientation found in the crystal structure.

The journey of Lab…………from 23rd Nov. to13th Dec,2016At Biology Training in TDC, IISC,Kudapura, Challakere

Pollen germination test represented by bud formation as observed Under 40X in Light Microscope

Pollen germinationEmploying

Catharanthus roseus

Pollens Initiation of bud formation

Maturation and elongation

of bud from pollen

SEED HEALTH TESTING

INFECTED SEED INFESTED WITH FUNGAL HYPHAE

Negative stainining

Catalase test

Stages of mitosis from Onion root tip

Bacterial Motility testing 3% KOH test for preliminary identification of Gram +Ve and

Gram –Ve Bacterial colony

Slide with mashed root tip after staining with acetocarmine

Principles of biology experiments meet the end at real life exposures …….

Pregnancy detection kit -

Sugar and protein estimation by urine detection kit

the presence or absence of hCG hormone in urine sample by immonodiffusionVISIPREG strip sandwich immune assay which uses anti-hCG inboth native and conjugated forms

Sugar normally is not found in urine. But when blood sugar level rise well above a target range which can occur in type 1 and type 2 diabetes, the kidneys sometimes releases sugar into the urine even when blood sugar levels are within a range

ODD assay in iimunotechnology

Different anitgen and antibody samples used for ODD assay in iimunotechnology

Ring of precipitation depends on conc. of antigen and antibody

Agarose gel

Distance of the wells leads to time dependent Precipitation ring development Antige

nAntibody Antibody

Determination of ESR count ESR is represented as fall of RBCs in mm per hour

Determination of heamoglobin conc. In blood in terms of percentage

Blood grouping Total count of WBC And RBC in different chambersOf Newbauer slide in Haemocytometer

Neutrophil

Basophil

Neutrophil

Lymphocyte

Monocyte Differential count of WBC by Giemsa staining

Thymus in dissected mouse

White transparent lobes above heart

Thymus, spleen and bonemarrow afterdissection

T cells and undiffrentiated lymphocyrte cells In bonemarrow extrafct after differential staining

WBC’s in thymus extract after differential staining

Ammonification in soil employing different soil samples

Control

Test

After 15 days Callus culture

Conductance of Callus culture

Amplified DNA after PCR

Purity checking of DNA after PCR in agarose gel electrophoresis

Development of peak employing MS software to check purity of amplified DNA PCR in progress

Polymerase chain reaction

Non amplified DNA as control

Bacterial growth curve Slide culture for study of autotrophs

Methyl Red test

Cell immobilization and inveratse assay from immobilized yeast cells on calcium alginate

SDS PAGE PROCESS AND DETERMINATION OF Mr FROM GRAPHICAL CALCULATIONS

Study of Barr Body from human cheek cells

Sterilized spatula

Staining of cheek cells (saliva samples)

Saliva sample from male – barr body absentAs one X chromosome absent

Sterilized spatula

Saliva sample from female – barr body presentAs two X chromosomes present

Barr body

Nucleus with no barr body

Nucleus

Barr body

Nucleus

Study of Drosophila, polytene chromosome and mitotic chromosome

Drosophila culturing

Chloroform application

Collected Drosophila after chloroform applied

Identification of male and female Drosophila

Microtomy of Drosophila 3rd Instar larvae for salivary gland dissection

Male and female Drosophila under Sterio Binocular Microscope at 4X

S…

Salivary gland

Neural Lobes ( frontal)

Poytene chromosome followed by saivary gland mashing and staining

Poytene chromosome

Mitotic chromosome followed by neural lobe mashing and staining

All cells are in metaphase stage

Qualitative estimation of Proteins

Quantitative estimation of Proteins

Lowry Method

Colorimeter

Quantitative estimation of carbohydrate

DNS Method

Study of protease activity from bromelein obtained form pine apple juice emplpying

gelatinase test

Control Test – Liquification of gelatin followed by incubation at 250C

Estimation of Microbial load on handscrubbing method

Antibiotic susceptibility test

Study of fungi

Colony of Rhodotorula an yeast Branched fungal hyphae

Estimation of chlorophyll

Agarose gel electrophoresis, restriction digestion, ligation of DNA

Pla B P R L

Pla- Plant DNA B- Bacterial DNAP- Plasmid DNA R- Restriction Digestion markersL- Ligated DNA

First test before initiation of the training (Answer key) date: 23.11.2016

Assignments based on each day lectures delivered by resource persons (key answers with questions)

Assignments based on each day lectures delivered by resource persons (key answers with questions)

Post training evaluation ( questions with key answers of test II)12.12.2016

ACKNOWLEDGEMENT

The training would not be successful even initiated by me

…

If I don’t thank the following persona…………..

TDC Training centre, IISC, Challakere for selecting me

1. Dr. B.S. Gowrishankar, HOD, Dept of Biotechnology,

SIT , Tumkur

2. Dr. Shivakumaraiah, Principal , SIT,. Tumkur and

Management of SIT, Tumkur

3. Faculty Staffs of Dept of Biotechnology, SIT, Tumkur

4. Non teaching staffs

5. My Family for support all the time invisibly

6. All my well wishers