Recovery studies and quality control

Inoculum preparation

Symptomatology

Gel documentation and image analysis

Production of DNas

Plasmid curing experiment

Gel documentation and image analysis

Voges Proskauer (acetoin production) test

Oxidative-Fermentative test

Oxidase test

Gram staining

Presumptive identification

Voges Proskauer (acetoin production) tes

Oxidative-Fermentative test

Oxidase tes

Gram staining

Presumptive identification

Sterilization

Determination of antileishmanial antibody responses in hamster

Preparation of media for bacterial cell culture

Cygwin

Cell culture

GFP-β-catenin-C (ARM)

Instruments

ELISA washing buffer

Immunization of the animals

Transformation buffer

RS-1 in Cancer samples

Single Cell Test

Single Cell Test

Primers used in this study

Waterto 3 mlTEMED 10 μlDenaturing (urea) sequencing gel (6%) composition10 X TBE 50 ml40% acrylamide 75 mlUrea 210 gm (7 M)Waterto 500 mlThis was filtered through a 0.45/0.22 μ milipore filter.For casting the gel 35 ml of the sequencing gel mixure was mixed with 150 μl10% APS and 25 μlTEMED

Formaldehyde agarose gel(For 50 ml)DEPC treated water 43 mlMOPS buffer 5.3 mlAgarose0.63 gmFormaldehyde2.6 mlThe above mix was boiled without formaldehyde to dissolve agarose and then at around 50ºC formaldehyde was added just before casting the gel.40% Acrylamide solutionAcrylamide39 gmBis-acrylamide 1 gmWater to 100 mlNon denaturing gel composition (50 ml)40% acrylamide solution 5 ml10 X TBE 5 mlH2O 40 ml10% APS 250 μlTEMED 30 μlSDS PAGE gel (12%)For resolving Gel (15 ml):30% Acrylamide solution 6 ml1.5 M Tris-Cl (pH 8.8)3.8 ml10% SDS150 μl10% APS 150μlWaterto 15 mlTEMED 10 μlFor stacking gel (3 ml):30% Acrylamide solution 500 μl1 M Tris Cl (pH 6.8) 380 μl10% SDS 30 μl10% APS 30 μl

Storage buffer for proteinTris-Cl (pH 8.0) 20 mMNaCl 300 mMDTT10 mMGlycerol 40 % Hybridization bufferTris-Cl (pH 8.0) 9 mMEDTA 0.35 mMSample buffer (for SDS-PAGE)Tris-Cl (pH 6.8) 150 mMSDS (20%) 6% v/vGlycerol 30% v/vβ-mercaptoethanol (5%) 15%Bromophenol blue 0.6% (w/v)EMSA binding bufferTris-Cl (pH 7.5) 10 mMNaCl 50 mMEDTA1 mMGlycerol 5 %DTT 5 mMDenaturing gel loading buffer with dyeFormamide 95%EDTA 20 mMXylene Cyanol 0.05 gmBromophenol blue0.05 gmNon denaturing gel loading buffer with dyeTris-Cl (pH 7.5) 250 mMBromophenol blue 0.02%Glycerol 20%

MOPS bufferMOPS 4.16 gm0.5 M EDTA 1.0 mlSodium acetate 0.68 gmWater (nuclease free) to 500 mlIt was filter sterilized and stored in an amber colored bottle. This was prepared as 10 Xstock solution and used at 1 X concentration.INOUE (PIPES) bufferPIPES (free acid) 10 mMCaCl2.2H2O15 mMKCl 250 mMMnCl2.4H2O 55 mMpH was adjusted to 6.7 with 1 N KOH.PIPES gets into solution when the pH is greater than 6.7. MnCl2was dissolvedseparately and added drop by drop with stirring. The pH was adjusted to 6.7 and filtersterilized and stored at –20ºC.Z buffer (for β-Galactosidase assay)Na2HPO416.1 gmNaH2PO45.5 gmKCl0.75 gmMgSO4.7H2O 0.246 gmβ-mercaptoethanol 2.7 mlWaterto 1000 mlpH was adjusted to 7.0 and stored at 4ºC.SDS running bufferTris-base 30.3 gmGlycine144 gmSDS 10 gmWaterto 1000 mlIt was prepared in 10 X concentration and diluted to 1 X for running

Citrate bufferCitric acid (0.1 M)4.7 volumeSodium citrate (0.1 M) 15.4 volumeTE bufferTris-Cl (pH 8.0) 10 mMEDTA 1 mMTBE bufferTris-Borate 90 mMTris-Borate 90 mMEDTA (pH 8.0) 2 mMThis was prepared as 10 X stock solution and used at 1 X concentration.TAE bufferTris-acetate 40 mMEDTA (pH 8.0) 2 mMThis was prepared at 50 X concentrated stock solution. Both TBE and TAE were usedas standard electrophoresis buffers

Buffers and solutions

Oligonucleotides/primers used in this study were designed using either free online-tool Primer3 (http://frodo.wi.mit.edu/) or Gene Runner software (http://www.generunner.net/). Oligonucleotides used in this study were commercially

synthesized from MWG Biotech Pvt. Ltd., Bangalore. All primers used in this study are listed in Table 2.3.Table 2.3: List of primers used in this study

Oligonucleotides

10mM EDTA0.1% SDS 1 M ureaToluidine blue staining solution:0.05% Toluidine blue20% Methanol2% GlycerolSolution was prepared in H2O.Destaining solution for polyphosphate gels:20% Methanol2% GlycerolSolution was prepared in H2O.Spheroplast buffer:50 mM Potassium phosphate (pH 7.5)0.6M Sorbitol0.2 X YPD mediumPS(PIPES-Sorbitol)buffer:10 mM PIPES-KOH (pH 6.8)200mM Sorbitol1 X protease inhibitor cocktail (Roche Cat # 04693159001)**To be added fresh before use

Citric-Phosphate buffer:0.5 M citric acid0.5 M dibasic sodium phosphatepH was adjusted to 5.0 with phosphoric acid and filter-sterilized.MES/TEA buffer:1 mM MES(2-(N-morpholino)ethanesulfonic acid)pH was adjusted to pH 5.0 with TEA(triethanolamine).Plasma membrane suspension buffer:50 mM Tris-HCl(pH 7.5)0.1mM EDTA0.1 mM Dithiothreitol 20% GlycerolPolyphosphate extraction buffer:50 mM HEPES (pH 7.2)

Other buffers

10 mM NaCl2.5 mM KCl10 mM MgCl210 mM MgSO4LB-ampicillin and LB-kanamycin platesLBmedium50 μg/ml ampicillin30 μg/ml kanamycinMedia and solutions were sterilizedeither by routine autoclaving at 121°C and 15 psi for 20 minor by filtration through membrane of 0.22 μm porosity

Luria Bertani (LB)0.5% Yeast Extract1% Tryptone1% NaClSuper Optimal Broth (SOB)0.5% Yeast Extract2% Peptone

Bacterial media

Yeast extract Peptone Dextrose (YPD)1% Yeast extract2% Peptone2% DextroseYeast Nitrogen Base (YNB)0.67% Yeast Nitrogen Base2% DextroseFor alternate carbon source utilization experiments, dextrose was replaced withother carbon sourcesviz.,sodium acetate, ethanol, oleic acid, glycerol and citric acid.Yeast Nitrogen Base (YNB) without ammonium sulphate and amino acids0.17% Yeast Nitrogen Base2% DextroseCasamino Acid (CAA)0.67% Yeast Nitrogen Base2% Dextrose0.6% Casamino acidsFor preparing plates, 2% agar was added tothe medium before autoclaving

Yeast media

Media

Nuclear extractionbuffer (without protease inhibitors)

Cytoplasmic extractionbuffer (without protease inhibitors)

For Cell fractionation

Transfer Buffer

Ethylenediamine tetraacetic acid (EDTA), pH 8.0ComponentsFinal concentrationFor 500 mlEDTA0.5M93.05gH2Oq.sThe pH is adjusted to 8.0 using 10M NaOH

6X DNA loading dye

Agarose gel

TAE

For DNA electrophoresis

(e) Stacking polyacrylamide gel

Wash buffer II10 mM Tris-HCI,pH 8.01 mM EDTA250 mM LiCl0.75% NP-400.75% sodium deoxycholateProtease inhibitor cocktailElutionbuffer II50 mM Tris-HCl,pH 8.0 10 mM EDTA 1% SDS

Lysis buffer50 mM HEPES,pH 7.5140 mM NaCl1% Triton X-1000.1 % sodium deoxycholate1 mM EDTAProtease inhibitor cocktail (added fresh)Wash buffer I50 mM HEPES,pH 7.5500 mM NaCl1% Triton X-1000.1 % sodium deoxycholate1 mM EDTAProtease inhibitor cocktail

Buffers for chromatin immunoprecipitation

0.5% Yeast Extract 1% Tryptone 1% NaClLB-ampicillin plates LB medium 100 μg/mL ampicillin Media and solutions were sterilized either by routine autoclaving at 121°C and 15 psi for 20 min or by filtration through membrane of 0.22 μm porosity.For yeast and bacterial growth, plates were preparedby adding 2% to the medium before autoclaving

Luria-Bertani (LB) medium forbacterialgrowth

Yeast synthetic complete medium without leucine(SC-Leu)0.67% Yeast Nitrogen Base without amino acids 76mg/L His76mg/L Ura76 mg/mL Trp76 mg/mL Met2% DextroseYeast sporulating medium1% Potassium acetate0.05% Dextrose

Yeast extract Peptone Dextrose (YPD)1% Yeast extract2% Peptone 2% Dextrose Yeast synthetic complete medium(SC)0.67% Yeast Nitrogen Base with amino acids 2% Dextrose1.92 g/LYeast Synthetic Drop-Out media supplement without Uracil76 mg/L uracilYeast synthetic complete medium without histidine(SC-His)0.67% Yeast Nitrogen Base without amino acids 1.92 g/L Yeast Synthetic Drop-Out media supplement without histidine2% DextroseYeast synthetic complete medium without uracil(SC-Ura)0.67% Yeast Nitrogen Base without amino acids 1.92 g/LYeast Synthetic Drop-Out media supplement without Uracil2% DextroseYeast synthetic complete medium without methionine(SC-Met)0.67% Yeast Nitrogen Base without amino acids 380mg/L Leu76 mg/L His76mg/L Ura2% DextroseYeast synthetic complete medium without tryptophan(SC-Trp)0.67% Yeast Nitrogen Base without amino acids 380mg/L Leu76mg/L His76mg/L Ura76 mg/L Met2% Dextrose

Yeast media(Media composition was followed as described by Sigma product data sheet)

Media

Liquid scintillation cocktail5 g PPO (2,5-diphenyloxazol)0.3 g POPOP (1,4-bis (5 phenyl 1,2-oxazole) Benzene Volume was adjusted to 1L with toluene.MUG (4-methylumbelliferyl β-d-glucuronide)extraction buffer1 mM MUG substrate50 mM Sodium dihydrogen phosphate (pH-7.0)10 mM EDTA0.1% Triton X-1000.1% Sodium lauryl sarcosine10 mM β-MercaptoethanolLactophenol solution (100 g)25 g Lactic acid (20.66 ml)25 g Phenol 50 g Glycerol (39.77 ml)These three components were mixed together and 1 volume of lactophenol was added to 2 volumes of ethanol

CAS solutiona) 0.06 g Chrome Azurol S dye in 50 mlb) Fe (III) solution: 10 ml1 mM FeCl310 mM HClc) 0.072 g HDTMA in 40 mlAll the above three solutions were mixed together and autoclaved prior to use

Other solutions

Transformation buffer I (Tfb-I) 30 mM Potassium acetate100 mM Rubidium chloride (RbCl2)10 mM Calcium chloride dihydrate (CaCl2.2H2O)50 mM Manganese chloride tetrahydrate (MnCl2.4H2O)15% (v/v) GlycerolpH was adjusted to 5.8 with 10% acetic acid and volume was adjusted to 500 ml with H2O. Transformation buffer II (Tfb-II) 10 mM MOPS75 mM CaCl2.2H2O10 mM RbCl2.2H2O15% GlycerolpH was adjusted to 6.5 with KOH (Potassium hydroxide) and volume was adjusted to 100 ml with H2O.

Buffers for E. colielectrocompetent cell preparation

50 mM Phosphate citrate buffer (pH-6.8)0.1M Citric acid0.2M dibasic Sodium phosphate16.9 ml Citric acid (0.1 M) and 33.1 ml Sodium phosphate (0.2 M) was mixed and volume was adjusted to 100 ml with H2O.Lipase assay0.1M Tris-HCl buffer (pH-8.2)pH was adjusted to 8.2 with HCl. 0.5 mM p-Nitrophenol standard solution8.69 mg p-Nitrophenol was dissolved in Tris-HCl buffer (0.1M) and volume was adjusted to 25 ml to make a final concentration of 25 mM.1volume of the above solution (25 mM) was diluted with 49 volume of 0.1 M Tris-HCl buffer to get a final concentration of 0.5 mM p-Nitrophenol standard solution.p-Nitrophenyl butyrate solution (420 μM)7.3 μl p-Nitrophenol butyrate (F.W. 209.2) 11 mg SDS650 μL Triton-X-100Volume was adjusted to 100 ml with H2O. Mixture was heated to 65°C in a water bath for 15 min, mixed well, and cooled down to room temperature prior to use. It can be stored upto 3 days at 4°C.Xylanase assay5 mg/ml RBB-xylan0.05 M di-Sodium hydrogen phosphate (Na2HPO4)

5 mg/ml RBB-Xylan was dissolved in 0.05 M Na2HPO4pH-7.5

Buffers for enzyme assaysCellulase assay

10% APS -30 μlTEMED -3 μlSDS loading buffer (2X)100 mM Tris-HCl (pH-6.8)20% (v/v) Glycerol4% (W/V) SDS0.02% Bromophenol Blue10% β-MercaptoethanolSDS-loading buffer was prepared as 2X stock solution in H2O and used at 1X concentration.SDS-PAGE running buffer14.4 g Glycine3.03 g Tris methylamine1 g SDSDissolved in H2O and volume was adjusted to 1L with H2O.Buffers for western blot analysisTransfer buffer (1 litre)14.4 g Glycine3.03 g Tris methylamine800 ml H2O 200 ml methanolBlocking and wash buffers (PBS-T)5% Fat-free milk0.05% Tween-20Volume was adjusted to 100 ml with1XPBS

Whole cell lysis buffer50 mM Sodium acetate 410 mg Sodium acetate anhydrous was dissolved in 80 ml H2O. pH was adjusted to 5.4 with glacial acetic acid and finally volume was adjusted to 100 ml with H2O.1 mM PMSF (phenylmethylsulfonyl fluoride) in isopropanol.Dialysis buffer50 mM Trizma basepH was adjusted to 7.5 by using concentrated HCl.Silver stainingFixing solution50% ethanol10% glacial acetic acid0.05% formaldehydeFinal volume was adjusted with sterile H2O.0.2% Silver nitrate solution (AgNO3)0.2 g AgNO3

0.075% formaldehyde (37% stock) Dissolved in 100 ml of H2O. Stored at 4°C for 1 hour in brown colored bottle.Developing solution 6% Sodium carbonate (Na2CO3)0.05% Formaldehyde (37% stock)0.02% Sodium thiosulphateStorage buffer50% EthanolSDS-PAGE30% Acrylamide solution29 g Acrylamide1 g Bis-acrylamideAcrylamide solution was prepared in H2O.Resolving gel mix (12%) (10 ml)H2O -3.3 ml30% Acrylamide:Bisacrylamide mix (29:1) -4 ml1.5 M Tris-HCl (pH-8.8) -2.5 ml10% SDS -100 μl10% Ammonium persulphate (APS) -100 μlN, N, N’,N’,-Tetramethylethylenediamine (TEMED) -4 μlStacking gel mix (5%, 3 ml)H2O -2.1 ml30% acrylamide:bisacrylamide mix (29:1) -500 μl1.5 M Tris-HCl (pH-6.8) -380 μl 10% SDS -30 μl

0.5% DEPC Added in H2O, stirred vigorusly and autoclaved prior to use.DNA sample loading buffer0.25% Bromophenol blue0.25% Xylene cyanol30% GlycerolDNA sample loading buffer was prepared in water

Buffers and solutions for protein extraction, analysis by SDS-PAGE (sodium dodecyl sulphate-polyacrylamaide gel electrophoresis) and silver staining

10 g of SDS (Sodium Dodecyl Sulfate) was dissolved in 80 ml of H2O, and volume was adjusted to 100 ml with H2O.CTAB/NaCl solution10% CTAB 0.7 M NaCl10 g of CTAB was dissolved in 80 ml 0.7 M NaCl solution by stirring it on a hot magnetic stirrer. Volume was adjusted to 100 ml with 0.7 M NaC1 solution.Lysozyme solution100 mg of lysozyme was dissolved in 1 ml of H2O (100 mg/ml).Proteinase K solution10 mg of proteinase K was dissolved in 1 ml of H2O (10 mg/ml).5 M Sodium chloride (NaCl) 292.2 g of Sodium chloride (NaC1; M.W. 58.44) was dissolved in 800 ml of H2O. Volume was adjusted to 1 liter with H2O. Sterilized by autoclaving.3 M Sodium acetate (NaOAc)(pH 5.2 and 7.0) 24.6 g sodium acetate anhydrous (CH3COONa; M.W. 82) was dissolved in 80 ml H2O. pH was adjusted to 5.2 with glacial acetic acid or 7.0 with dilute acetic acid. Volume was adjusted to 100 ml with H2O. Sterilized by autoclaving.Phenol:Chloroform:Isoamyl alcohol (25:24:1) solution25 ml Tris-equilibrated phenol24 ml Chloroform1 ml Isoamyl alcoholDEPC (diethyl polycarbonate) treated water

50 mM Tris-HCl (pH 8.0)10 mM EDTA (pH 8.0)100 μg/ml RNaseVolume was adjusted to 100 ml with sterile H2O.10% SDS

Buffers and solutions for extraction and analysis of genomic DNA and RNAResuspension buffer (P1)

PBS was prepared as a 10X stock solution and used as a 1X concentration.Tris-HCl buffer0.5 M Trizma BasepH was adjusted to 7.6 using concentrated HCl.Tris-Cl buffer was prepared as a 10X stock solution and used as 1X concentartion.Tris-EDTA (TE) buffer10 mM Tris-HCl (pH 8.0)1mM EDTATris Acetic acid-EDTA (TAE) buffer40 mM Tris base0.5 M EDTApH was adjusted to 8.5 with glacial acetic acidTAE buffer was prepared as a 50 X stock solution and used at 1 X concentartion.Potassium Phosphate buffer (0.1 M)1 M Potassium phosphate dibasic (K2HPO4)1 M Potassium phosphate monobasic (KH2PO4)61.5 ml of 1 M K2HPO4was mixed with 38.5 ml of 1 M KH2PO4, pH was adjusted to 7.0 and volume was adjusted to 1 L with H2O

Phosphate-Buffered Saline (PBS)137 mM NaCl2.7 mM KCl10 mM Na2HPO42 mM KH2PO4pH was adjusted to 7.3 before autoclaving

Common buffers

Buffers and solutions

Casamino acid (CAA)0.67% Yeast Nitrogen Base2% Dextrose0.6% Casamino acid

Yeast Extract-Peptone-Dextrose (YPD)1% Yeast Extract2% Peptone2% DextroseYeast Nitrogen Base (YNB)0.67% Yeast Nitrogen Base2% DextroseFor alternate carbon source utilization experiments, dextrose was replacedwith other carbon sources viz.,ethanol, glycerol, oleic acid and sodium acetate.Ethanol, oleic acid and sodium acetate were used at afinal concentration of 2%and glycerol was used at a final concentration of 3%

Yeast media

2.5 mM KCl10 mM MgCl210 mM MgSO4SOCSOB mediumwas modified to prepare the SOC medium.20 ml of sterile 1 M glucose solution was added to the autoclaved SOB medium to obtainafinal concentration of 20 mM glucosein 1 litre of medium.AntibioticsAmpicillin 60 μg/mlKanamycin 30 μg/mlStock solution of antibiotics (50 mg/ml) were prepared in sterile water. Prior to storage at -20°C,antibioticswere filter sterilizedthrougha0.22 μm membrane filter. Before pouring the plates,antibiotics were added to moderatelywarm LB-agar medium

Luria Bertani (LB)0.5% Yeast Extract1% Tryptone1% NaClSuper Optimal Broth (SOB)0.5% Yeast Extract2% Peptone10 mM NaCl

Allmedia and solutions were sterilizedby autoclaving at 121°C and15 psi for 20 min.For preparation of plates,2% agar was added to the medium before autoclaving.Heat labile components and reagents were filter sterilizedby passing them through a 0.22 μm membrane filter

Bacterial media

Media

Antibodies

Table 2.3: Antibodies used in this study