The proteins were resolved using denaturing SDS-PAGE gel and after completion of the run, the gel was over laid on a nitrocellulose paper cut to the size of gel and kept in the blotting cassette in the presence of blotting buffer. Finally the cassette was put in the mini transblot apparatus (Bio Rad) and blotting was done for 4 hours at a constant voltage of 60 V. Then the membrane was taken out and rinsed in PBS containing 0.1% Tween - 20 (PBST) for 5 minutes by gentle shaking. Later the membrane was immersed in 5% non-fat milk solution in PBST with gentle shaking for 1 hour at 37°C. The membrane was washed off from the traces of the fat free milk with PBST and the membrane was over laid with primary antibody diluted in PBST for 3 hours at 4°C with shaking. After incubation the membrane was washed with PBST and layered with

Immunobloting

f. 5 μl of water was then spotted on each spot for 30 sec and removed using Whatman filter paper strips. This step was repeated once. g. 1-2 μl of SAP matrix was then applied to each spot and allowed to dry. h. The chip was then placed in the SELDI machine

a. 5 μl of 10 mM HCl was added to each spot on the chip and removed after 5 min. using Whatman filter paper strips. b. Washing was given by spotting 3 μl of water for 30 sec on each spot followed by removal using Whatman filter paper strips. This step was repeated two times. c. 10 μl of low stringency/ high stringency buffer was then added to the spot and kept in humid chamber for 5 min. followed by removal using Whatman filter paper strips. d. 3 μl of sample prepared in low stringency/ high stringency buffer was then added to the spot and incubated in humid chamber for 30 min. e. Washed the spot with 5 μl of low stringency buffer/ high stringency buffer/ buffer of pH 3.0/ pH 5.0/ pH 7.0 for 30 sec and removed using Whatman filter paper strips. This step was repeated five times.

Activation of CM10 (weak cation exchange ) array

Fresh overnight culture of the E. coli strain (DH5α) was subcultured 1:100 in 250 ml LB/SOB media at 18 ̊C and 2500g and allowed to grow to an A600=0.55. Culture was chilled on ice and centrifuged at 2500g at 4 ̊C for 10 min. The cell pellet was redissolved in 80 ml ice-cold Inoue transformation buffer (55 mM MnCl2, 15 mM CaCl2, 250 mM KCl, 10 mM PIPES pH6.7). This cell suspension was centrifuged at 2500g at 4 ̊C for 10 min. and the cell pellet was resuspend in 20ml ice-cold Inoue buffer with 1.5 ml DMSO. This mixture was then placed on ice for 10 min. Aliquots of this suspension were dispensed into chilled, sterile microfuge tubes that were snap-frozen in a bath of liquid nitrogen. Tubes were stored at ─70 ̊C until required. For transformation the cells were thawed on ice and plasmid DNA was added followed by the standard transformation protocol

Preparation of ultracompetent cells

PonceauS stain Instant Blue (Biorad)

Protein loading dye (6X) Tris-Cl (pH 6.8) 300 mM SDS 12% (w/v) Bromophenol blue 0.6% (w/v) Glycerol 60% (v/v) 600 mM β-mercaptoethanol

Stains and Dyes

C-1 (5,5' ,6,6' -tetrachlorol,1' ,3,3' tetraethyl benzimidazoly 1 carbocyanine iodide) is an anionic mitochondrial vital dye (10mm stock prepared in DMSO) that is lipophilic and becomes concentrated in the mitochondria in proportion to the membrane potential; more dye accumulates in mitochondria with greater potential and ATP generating capacity. The dye exists as a monomer at low concentrations that emit a green fluorescence (530nm) but at high concentrations forms J aggregates that emit red fluorescence (590nm). The ratio of the two fluorescences gives a ratiometric comparison of mitochondrial membrane potential. Following appropriate treatment, 107 (in 1mL medium) cells were transferred to an MCT containing 10pL of the working stock (0.4mM ) of the dye (final concentration of 4pM), and incubated at 37°C for exactly 10 min in the dark. This was followed by centrifugation at 1811 x g for 3 min at RT. The pellet obtained was resuspended in M199 medium containing 10% FBS and centrifuged at 1811 x g for 3 min at RT. Two more such washes were given, after which the pellet was resuspended in 2mL M199 + 10% FBS and fluorescence measured at 485nm/ 530nm and 535nm/ 590nm

Assay for measuring Mitochondrial Membrane Potential (JC-1 Staining)

Germany) as per manufacturer's protocol. Briefly, the gel was solubilised by incubating it with buffer QG (composition proprietary) at S0°C for 10 min. The solubilized gel was loaded onto a binding column and centrifuged at 12000 x g for 1 min. The flow through was discarded and the column was washed once with buffer PE containing ethanol. The DNA bound to the column was eluted using the elution buffer provided with the kit, or alternatively with nuclease-free water. The concentration of the obtained DNA was estimated by measuring the absorbance at 260nm (A26o) and using the known formula: DNA concentration= A26o X SOX dilution factor.

To elute DNA from agarose gel, samples were loaded on a low-melting agarose gel. The samples were resolved and visualized under UV transilluminator, and the band of interest was excised quickly using a scalpel blade. The volume of gel slice was quantified by weighing and the DNA eluted using MinElute Gel Extraction kit from Qiagen (Hilden

Elution of DNA from agarose gel

Band intensities in gel autoradiograms were determined by densitometry with the aid of the Fujifilm Multi Gauge V3.0 imaging system.Equal areas of radioactive bands (preferably the unbound probe) were boxed and the PSL (Photostimulated luminescence) valueswere further considered. For Kd(dissociation constant)calculations, the values thus obtained for each lane were expressed as a percentage with respect to the PSL for the lane without any protein taken as 100%

Densitometry

Plasma membrane H+-ATPase activitywas measured inthe total membrane fraction as described previously (Nakamura et al., 2001).5μg totalmembrane fraction was incubated at 30 ̊C in 120 μl reaction buffer containing 10 mM MgSO4and 50 mM KCl in 50 mM MES (pH 5.7) with 5mM adenosine tri-phosphate (ATP). To eliminate possible contribution of residual ATPases, viz.,vacuolar ATPases, mitochondrial ATPases or non-specific phosphatases, 50mM KNO3, 5mM NaN3and 0.2mM ammonium molybdate were used, respectively, in the assay mixture. Reaction was stoppedafter 30 minby adding 130μl stop-developing solution containing 1% (w/v) SDS, 0.6M H2SO4, 1.2%(w/v)ammonium molybdate and 1.6%(w/v) ascorbic acid. Amount of inorganic phosphate (Pi) liberated was measured at A750nmafter 10 minincubation at room temperature. A standard curve prepared with0-50 μmolesof KH2PO4 was used fordetermination of total Piamount.ATPase activity of the plasma membrane was expressed in micromoles of Pireleased per milligram protein per min. ATPase activity was also determined in the presence of plasma membrane H+-ATPase inhibitor diethylstilbestrol (DES,Sigma# D4628),wherein total membrane fraction was incubated with 0.2mM DES for 5 min, prior to the enzymatic measurement

Plasma membrane H+-ATPase activity assay

dithiothreitol and1X protease inhibitor cocktail. Cell suspension was rapidly frozen at -80 ̊C,thawed and lysed with 0.5mm acid-washed glass beadsin a homogenizer (FastPrep®-24,MP Biomedicals)at maximum speed of 60 secfive times. Homogenate wasdiluted with 5mlTris-HCl (0.1M; pH 8.0)solutioncontaining 0.33M sucrose, 5mM EDTAand 2mM dithiothreitoland centrifuged at 1,000g for 3 minat 4 ̊C. Supernatant was collected and centrifuged again at 3,000g for 5 minat 4 ̊C to remove unbrokencells. The resulting supernatant was centrifuged at 19,000g for 45 minat 4 ̊C to obtain total membrane fraction. Total membrane pellet was resuspendedin 100μl membrane suspension buffer and stored at -80 ̊Ctill further use. Total protein concentration in the membrane fraction was estimated using BCAprotein assay kit (Thermo Scientific, US) with bovine serum albumin (BSA) used as astandard

Isolation of total membrane fractions from C. glabratastrains were carried out as described previously (Fernandes et al., 1998). Cells grown to log-phase under different environmental conditionswere harvested, washed and suspended to afinal density of 20 OD600cells in 1 ml solution containing100mM Tris (pH 10.7),5mM EDTA,2mM

Total membrane preparation

To assess the activity of plasma membrane proton pump, CgPma1, in cells grown in differentexternal pH environment,whole cell acidification assaywas carried out.This assay is a measurement of glucose-responsive proton pump activityin live cellsand is based on a decrease inthe pH of a weakly-buffered solutionupon extrusion of H+ions from thecell. The amount of change in the pH of the medium represents a crude measurement of the activity of functional plasma membrane proton pump in live cells. Whole cell acidification assay was conductedwithcellsgrown in YNB pH 5.5 and YNB pH 2.0medium as described previously (Martinez-Munoz and Kane, 2008) with slight modifications.After growth at30 ̊C for 2 h, cells were harvested, washed and resuspended(1.5-3.0 mg wet weight/ml) in 15ml MES/TEA (1mM; pH 5.0) buffer. Cell suspension was kept at 25 ̊C with continuousagitation. Extracellular pH of the buffer solution was recorded at 1 mininterval for 20 minwith the help of a pH meter(BT-600, BoecoGermany). To activate plasma membrane proton pumping, glucose and KCl were added to a final concentration of 40mM after 3 and 8 minincubation, respectively. Plasma membrane proton pump activitywas plotted as a change in the pH of the extracellular solutionversustime

Whole cell acidification assay

Measurement of plasma membrane H+-ATPase activity

A single colony of E.coliBW23473strainfrom a freshly-streaked LBplate was inoculated in50ml LB medium. Culture was incubated overnight at 37°C with shaking at 200rpm. 25ml of the overnight-grown BW23473 culturewas transferred to500ml pre-warmed LB medium andincubated at 37°C till the OD600reached to 0.4. After incubation, cultureswere transferred to an ice-water bathandcentrifugeat 1,000g for 15 minat 4°C. Cells were washed twice with 500ml ice-coldwater, thrice with250ml ice-cold 10% glycerol solution and resuspendedin 1ml 10% glycerol solution. Cell suspension wasnormalized to final cell densityof 3x 1010cells/ml and dispensed in 50μl volume into sterile ice-cold microcentrifuge tubes. Aliquots weresnap frozen inliquid nitrogen and stored at -70 ̊C for further use

Electro-competentcell preparation

5-10 ml saturated bacterial culture harboring the desired plasmid was harvested at 5,000 g for 3 min. Plasmid DNAwas isolated using QIAprep Spin Miniprep Kit (Qiagen, USA) or GenElute™ HP Plasmid Miniprep kit (Sigma-Aldrich, USA) as per manufacturer’s instructions

Bacterial plasmid isolation

temperature. Genomic DNA pellet was dissolvedeitherin 50 μl 0.1X TE or molecular biology grade water containing 0.3 μlAmbion RNAse cocktail and incubated at 37ºC for 30 min.After RNA digestion, 100 μl of 0.1X TE or nuclease-free water was added to the tube and stored at -20ºC. Quality of extracted genomic DNA was checkedon 0.6% agarosegel by electrophoresis

Desired C. glabratastrain wasgrownovernight in YPD liquid medium and yeast cells were harvested by centrifugation at 2,500g in 15 ml polypropylene tube.Yeast cells were washed with PBS, resuspendedin 500μl lysis buffer (Buffer A) andwere transferred toa2ml microcentrifuge tube. Yeast cells were incubated for 15 minon a thermomixer set at 65 ̊C and 750 rpm. After incubation, 0.5 gm glass beads (0.5 mm) and 500 μl PCI solution were added to thetube. Yeast cells were lysedthree times for 45 seconds each on a bead beating apparatus with intermittent cooling on ice to prevent overheating. Cell lysates were centrifugedat 7,500gfor 5 minandupperaqueous phase (300-350 μl) wastransferred carefully to a new 1.5 ml microcentrifuge tube. 1 mlabsolute ethanol was added andmixedwellby inverting the tube3-4 times. To precipitate genomic DNA, suspension was centrifuged at 7,500g for 10min.Precipitated genomic DNA was washedwith 70% ethanolanddried at room

Genomic DNA isolationby glass bead lysis method

For opsonization,C. glabratacells were incubatedwith 1 μg/μl human IgG for 30 min at 37°C and washed thrice with PBS. Alternatively, yeast cells were incubated with 25% human serum at 37°C for 30 min followed by threePBS washes

Opsonizationof C. glabratacells

undertissueculture conditionsfor 45-60min andfixed in 3.7% formaldehydeas described earlier.For DAPI staining, Vectashield mounting medium containing DAPI was used and slides were visualized under confocal microscope.For heat killing, yeast cells were harvested from 1 ml culture, washed, resuspended inPBS andwere incubated at 95°C for 5 min

PMA-treated THP-1 macrophages were infected with C. glabratacells to a MOIof 1:1 in four-chambered slides and incubated at 37°C and 5%CO2. After 1 hcoincubation, each chamber was washed thrice with PBS to eliminate extracellular yeast cellsand medium was replaced with fresh prewarmed RPMI medium containing100 nM Lysotracker Red DND-99.Infected THP-1 macrophageswere incubated

Lysotracker staining

Themixture is incubated in a water bath at 37⁰C for 15 min and afterwards transferred on ice and 4μl of DNA loading buffer is added. The samples were then run on a polyacrylamide gel electrophoresis which had been pre-run for 30 min. Electrophoresis was carried out at 4⁰C for 3h till the bromophenol blue migrated to 2cm above the bottom of gel. The gel was taken out and kept on Whatman filter paper sheet and covered by saran wrap followed by drying in a gel dryer at 80⁰C for 1h under suction. The dried gel was exposed to phosphoimager screen by keeping in phosphoimager cassette overnight

A binding reaction mixture was prepared by adding the following components to a microcentrifuge tube on ic

Binding reaction

The reaction was carried out by incubating at 37⁰C for 30 min. The reaction was stopped by adding 2μl of 0.5M EDTA, pH 8.0 and keeping on ice. A spin column was prepared using 1ml syringe and packed with sterile Sephadex G50 slurry and reaction mixture is applied on the top. The eluate is collected in different microcentrifuge tubes and radioactivity was counted using Geiger counter. The tube showing 7 to 9X106was used for experiment. The column containing the unincorporated [γ-32P] ATP was discarded in radioactive waste bin. The radiolabelled oligonucleotides were annealed with their corresponding complementary unlabelled oligonucleotides. A 50 fold molar excess of the latter was used for annealing for conversion of labelled single strand to double strand. Thetubes were kept in boiling waterbath for 3 min followed by room temperature for 30 min. The tubes were transferred to ice and the oligonucleotides were diluted to 4fmoles/μl using sterile H2O

The oligonucleotides were labelled at their 5'end with 32P using T4 polynucleotide kinase (T4 PNK) enzyme in a reaction given belo

end labelling of the oligonucleotides

Electrophoretic mobility shift assay

DNA loading dye

Agarose gel

TAE

For DNA electrophoresis

Stripping Buffer

Leupeptin

in 5% fat free milk solution in TBST (1:7000) for 45 min at room temperature and then washed thrice.The detection of signal was performed with ECL detection reagent (Amersham Biosciences) followed by detection of signal either on X-ray film (Hyperfilm-ECL, Amersham Biosciences)or in a chemidoc system (Proteinsimple, California, USA).The blot was reprobed with anti-tubulinor anti-GAPDHantibody to ensure equal loading of extracted protein

Materials and Methods472.2.7 Estimationof protein concentration in cellular lysatesBradford method(Bradford, 1976)was used to determine the quantity of protein in various samplesin a 96-well plate. Bradford’s reagent was prepared by diluting Bradford dye with water in the ratio of 1:5.For estimating the concentration of protein in a particular sample, 50μl volume reactionwas set and200μl of freshly prepared Bradford’s reagent was added. The complex givesa purplish colorwhose intensity is proportional to the amount of protein present in the sample. A standard curve was also generated using increasing concentrations of BSA (50 μg/ml, 100 μg/mland 200μg/ml).Cell lysatesof test samples werediluted to1:50 in the same volume.Each sample (including blank and standards) was taken in duplicates.The concentration of protein was measuredusing the ELISA reader at 570 nm. The unknown protein concentration (X) was calculated as follows:where,OD1& OD2: Optical densities of Standard (Std) 1 & Standard (Std) 2, respectively.BSA: Bovine serum albuminX×50 (dilution factor)/1000 = YConcentration of unknown protein (μg/μl) = Y × OD2.2.8 Immunoblotting(Western Blotting)Immunoblotting was performed as essentailly described by Lee (Lee, 2007). Equal amounts of protein were resolved on a denaturating SDS-polyacrylamide gel (8-12%). After completion of the run, the gel was transferred onto PVDF membrane and placed in the blotting cassette. The cassette wasthenput intothe mini transblot apparatus and transfer was done for 2-3 hours at a constant voltage of 80 V, depending on the size of the protein. Post transfer, membrane was rinsed in TBS containing 0.1% Tween-20 (TBST) and blocked with 5% non-fat milk in TBST for 1 h at 37ºC,on a gentle shaking rotator. After blocking, membrane wasrinsed thrice in TBST and incubated with primary antibodydiluted in TBST (ranging from 1:1000 to 1:10000, depending upon antibody used) for either3h at room temperature or overnight in the cold room.The membrane was then washed thrice with TBST and incubated withhorseradish peroxidase(HRP)-conjugated secondary antibody diluted

Immunoblotting(Western Blotting)

Extraction buffer

MTT reagent

For Cytotoxicity assays

Blocking buffer

Yeast were grown till mid-log phase 0.6-0.8 OD600in an appropriate medium and 10 mL of culture was pelleted at 2500 x g. Cells were suspended in 350μLof AEbuffer,mixed with 50μLof 10% sodium dodecyl sulphate and 400μLacid phenol(pH4.3) and immediately shaken vigorously on a dry bath (Eppendorf)at 65°C for 15 min.The tube was then quickly chilled on iceand centrifuged at 12000 x gfor 15 min to separate the aqueous phase from the phenol. After centrifugation, the aqueous phase was transferred to a new tube and extracted with an equal volume of chloroform.RNA was precipitated by adding 50 μLof 3 M sodium acetate (pH 5.3) and equal volume of 100% ethanol followed by incubation at -20°C for 2 h, andcentrifugation at maximum speed for 30 min at 4°C. The pellet obtained was washed in 70% ethanol, dried at room temperature anddissolved in an appropriate volume of DEPC-treated water. The concentration of RNA was estimated by measuring A260using a Nano Drop Spectrophotometer (ND1000). To monitor different classes of rRNA levels, 10 μg of total RNA from each strain was resolved on a 1.2% formaldehyde-agarose gel

RNA extraction by hot-phenol method

0.83mL1.5 M Tris-HCl,pH 6.8 50μL10% SDS 50μL10% Ammonium persulfate (APS)8 μLN,N,N′,N′-Tetramethylethylenediamine (TEMED)Resolving gel mix (12%) (20 ml)6.6 mLH2O 8 mL 30% acrylamide:bisacrylamide (29:1) mix 5 mL1.5 M Tris-HCl,pH 8.8 200 μL10% SDS 200 μL10% Ammonium persulfate (APS)8 μLN,N,N′,N′-Tetramethylethylenediamine (TEMED)

Whole cell lysis buffer for yeast (Homogenizing buffer) 50 mM Tris-HCl,pH 7.52 mM EDTA yeastprotease inhibitor cocktail SDS-PAGE 30% Acrylamide solution 29 g acrylamide 1 g bis-acrylamide dissolved in 100 mLH2O. 10% sodium dodecyl sulfate (SDS) 10 g SDS in 100 mLH2O Stacking gel mix (6%)(5 mL)3.4mLH2O 0.63mL 30% acrylamide:bisacrylamide (29:1) mix

Buffers for SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis)

final supernatant, beads were boiled in the equal volume of 2X SDS loading buffer (Lamelli Buffer) for 5 min. at 95°C. Bound protein complexes were collected by brief centrifugation (1 min.) at 14000 RPM and the supernatant containing the eluted protein fraction was resolved in SDS gel and analyzed by western blotting technique. For denaturing immunoprecipitation, cells were harvested in 1X PBS andpelleted. Cell pellet was resuspended in denaturing lysis buffer (50mM Tris-HCL, 100mM β-mercaptoethanol, 1% SDS, 5mM EDTA,0.5mM PMSF, 1 mg/ml aprotinin and 1 mg/ml pepstatin) (200μl for 100mm culture dish) and boiled for 10 min at 99°C. Cells suspensionwas briefly sonicated. Ice-cold 1X NETN (800μl) was added to the suspension after sonication and incubated on ice for 20min.,cells were centrifuged at 14000 rpm for 10 min. The supernatant was collected and used for immunoprecipiation by following the standard protocol

Protein-A or protein G beads (50 μl of 50% agarose beads) were washed twice with NETN lysis buffer. 2-5 μg of specific antibody was added to beads (in 1ml NETN) and were incubated with beads for 1 h at 4°C on a rotary shaker. Beads were collected by spinning at 500 g for 2 min.and supernatant was removed. 200-600 μg of totalprotein (mammalian cell lysate or bacterial cell lysate) was added to the beads and incubated for 2 hrs at 4°C on a rotary shaker. Beads were washed four times with the lysis buffer, each time by centrifuging at 500 g for 2 min at 4°C. After discarding the

Immunoprecipitation

ForEPS isolation,X. oryzaepv. oryzicolastrains were plated on PS agar plateand incubated at 28°C. Bacterial lawn was dissolved in 15 ml 1X PBS and 100 μl formamide, and centrifuged at 12,000 g for 6-8 min at RT. Before centrifugation, 1 ml cell suspension was diluted, and plated to get the CFUs. For EPS precipitation, 250 ml chilled acetone was added to the supernatant, and kept at 4°C for overnight (Dharmapuri and Sonti, 1999). EPS was pelleted down at 7000 g for 10 min at 4°C, washed with 10 ml acetone, and kept for drying. After drying, it was dissolved in appropriate volume of water, and quantitated by colorimetric method for estimation of pentoses and hexoses by phenol-sulphuric acid method (Dharmapuri and Sonti, 1999)

EPS isolation and estimation

For DNA precipitation after digestion, 500 μl nuclease free water was added to the digested DNA fragment. Equal volume of phenol:chloroform:isoamyl alcohol (25:24:1) was added to the mixture and centrifuged at 13,000 g for 10 minat RT. Upper aqueous phase containing DNA fragment was transferred to fresh microcentrifuge tube and DNA was precipitated by adding 0.7 volume of iso-propanol and 1/10thvolume of sodium acetate. Precipitated DNA was washed with 70% ethanol, pellet was air dried for 20-30 min at RT and dissolved in nuclease-free water

DNA precipitation

A microtipful cells of bacterial strain from appropriate medium was resuspended in 20 μl sterile water and incubated at 98°C for 10 min for cell lysis. 2 μl of heat-lysed cell suspension was used as template in 25 μl PCR reaction

Xanthomonasand E.colicolony PCR

The tannin sample (1.0 ml) was added to 2.0 ml BSA solution in a 15 ml glass centrifuge tube. The solution was mixed and allowed to stand at room temperature for 15 min and then centrifuged at 10000 rpm for 15 min to separate the precipitated tannin-protein complex as pellet. The supernatant was discarded and the pellet and the walls of the tube were washed with acetate buffer without disturbing the pellet. Now, the pellet was dissolved in 4.0 ml of SDS-triethanolamine solution and to this, 1.0 ml of ferric chloride reagent was added and was mixed immediately. After 30 min of addition of ferric chloride, the absorbance was noted at 510 nm on spectrophotometer. All observations were carried out in triplicates. The concentration of the tannin was determined with the help of tannic acid (Sigma) standard curve prepared in the range of 0.2 to 1.0 mg/ml

The procedure of Hagerman and Butler (1978) was used to estimate the tannin content in different tannin sources. Reagents: Bovine serum albumin (BSA) 1.0 mg/ml: 10.0 mg of bovine serum albumin was dissolved in 10.0 ml of 0.2 M acetate buffer, pH 5.0, containing 0.17 M sodium chloride. Sodium dodecyl sulfate (SDS)-triethanolamine solution: The solution contained 1.0% SDS and 5.0% (v/v) triethanolamine in distilled water. Ferric chloride reagent (0.01 M): 1.62 g of ferric chloride was dissolved in 1.0 L of 0.01 N hydrochloric acid.

Tannin estimation (Hagerman and Butler, 1978)

To perform restriction digestion of plasmid DNA and PCR-amplified DNA products, restriction enzymes were procured from NEW ENGLAND Biolabs(NEB). Restriction digestion was set in 50 μl reaction volume with appropriate buffer and 1X BSA. For ligation of DNA fragments obtained after restriction digestion, T4 DNA Ligase enzyme (NEB, M0202M) was used. All ligation reactions were set in 20 μl reaction volume containing 1X ligase buffer, 3-10 units of DNA ligase enzyme and vector to insert molar ratio of 1:3. The ligation mixture waseitherincubated at 16°C for 16h or at room temperature for 2-3 h. Post incubation, ligation reaction was inhibited by heatingtubes at 65°C for 15-20 min.2-5 μl of ligation mixture was used to transform ultra-competent E. coliDH5αcells

Restriction digestion and ligation

To perform restriction digestion of plasmid DNA and PCR-amplified DNA products, restriction enzymes were procured from NEW ENGLAND Biolabs(NEB). Restriction digestion was set in 50 μl reaction volume with appropriate buffer and 1X BSA. For ligation of DNA fragments obtained after restriction digestion, T4 DNA Ligase enzyme (NEB, M0202M) was used. All ligation reactions were set in 20 μl reaction volume containing 1X ligase buffer, 3-10 units of DNA ligase enzyme and vector to insert molar ratio of 1:3. The ligation mixture waseitherincubated at 16°C for 16h or at room temperature for 2-3 h. Post incubation, ligation reaction was inhibited by heatingtubes at 65°C for 15-20 min.2-5 μl of ligation mixture was used to transform ultra-competent E. coliDH5αcells

Restriction digestion and ligation

were mixed or shaken every 15 min during the incubation as to keep them in suspension. At the end of the incubation, cells were washedagain with 800 μLof rinse bufferby centrifugation at 500 x gfor 6 min and the supernatant was aspirated. BrdU incorporation was detected by adding 100 μLof antibody staining solution containing 5 μLAlexa488 dye labeled anti-Brdu antibody with 95 μLof rinse buffer(1:20 dilution), and the cell suspension was incubated for 30 min at room temperature in the dark.At the end 350 μLof PI staining buffer was added to each sample and incubated for an additional 30 min at room temperature in the dark. Cells were analyzed forthe presence of DSBs by flow cytometry on a FACS ARIA instrument (BD). Viable cells were analyzed for the presence of DNA DSBs by excluding the hypodiploid (<G0/G1)population, using FACS DIVA software (BD). Medianfluorescence values of the treated cells were plotted as a fold difference over untreated controls, using GraphPad Prism 5

Cells were grown in 35mm dishes at 30% initial confluence. At 60% confluence, cells were treated with 0.2 mMhydroxyurea for 12 h. After treatment,media containing drug was removed and fresh media was added for recovery of the cells from genotoxic stress.DNA DSBs were monitored during the treatment period and recovery period of 3, 6, 9 and 12 h. At each time point cells were harvested and fixed using 70% icecold ethanol by gentle vortexing at very low speed and kept overnight at -20ºC. A TUNEL assay to detect DSBs was conducted using Apo-DirectTunel assay kit (A35127, Invitrogen). After overnight fixation, cells were washed with 800 μLof wash buffer, and 50 μLof DNA labeling solution [10 μL reaction buffer, 0.75 μL of TdT enzyme, 8 μL of BrdUTP and 31.25 μLof dH2O (Sigma)]was added to each sample, incubated at 37ºC for 4 h. Samples

Detecting DNA DSBs by TUNEL assay

Cultures in mid-exponential phasenormalized using A600and solubilizedin 1X sample buffer at 99°C for 5 min were subjected to electrophoresis on 12% sodium dodecyl sulfate (SDS) -polyacrylamide gels. Cell extracts equivalent to 0.04A600(1X) and 0.02A600(0.5X) were loaded and run using Tris-glycine-(SDS) buffer. Separated proteins were electrotransferred to PVDF polyvinyledene difluoride) membrane (Amersham) electrophoretically by a semi-dry method using Bio-Rad apparatus.The transfer was done for 2-3 hrs using a voltage of 75V at 4oC and membrane was probed using anti-FtsZ primary antibody at 1:5000 dilution (rabbit, polyclonal), washed and probed with anti-rabbit IgG conjugated to horseradish peroxidase (HRP) at 1:20000 dilution, as described(Sambrook & Russell, 2001).Membranes were developed with chemiluminescencereagent (Amersham ECL Prime) and visualized with the aid of a chemiluminescence detection system according to the manufacturer’s protocol (Sigma Chemical Co., St. Louis, MO). Quantification of band intensity and subtraction of background was done using Fujifilm Multi Gauge V3.0 imaging system(Image Quant software)

Gel Electrophoresis and Western blotting

Growth curves were generated to compare the growth rates of E. coli test strains with control strains manually. The appropriate dilutions of the overnight cultures in desired media were made and allowed to grow at required temperature till faint turbidity was visible. At this point samples were collected every 30 minutes until stationary phase was attained. The growth curves weregenerated using Microsoft Excel or SigmaPlot software and growth rates were calculated from the slope of the graph which, in turn, was used to calculate generation time

Estimation of growth rates

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UBE2A

UBE2A

foolish imaginations of his heart

The first color image of the earth, taken by the Apollo 8 astronauts in 1968

Apollo 8 was the first moonshot. No human being had ever been beyond low Earth orbit. Even the Apollo 8 astronauts — Frank Borman, James Lovell Jr. and Bill Anders — struggled to wrap their heads around what they were about to do.

In the State of New York even the right of transit for a slave has been denied by her tribunals; and the States of Ohio and Iowa have refused to surrender to justice fugitives charged with murder, and with inciting servile insurrection in the State of Virginia. Thus the constituted compact has been deliberately broken and disregarded by the non-slaveholding States, and the consequence follows that South Carolina is released from her obligation.

The moveBW project offers drivers an attractive option that links motorized personal transport with alternative modes of transportation. An easy-to-use mobility assistant on your smartphone helps you choose a mode of transportation and reliably guides you to your destination. Users of the mobility assistant can book different types of transportation – yet receive just one bill that lists every mode booked during the past month. To plan intermodal routes, the mobility assistant considers services such as public transportation, car sharing, bike sharing, and parking-space management as well as information on traffic jams and construction areas. MoveBW encourages people in the greater Stuttgart area to efficiently utilize all modes of transportation, which eases congestion. This project also aids local authorities in optimizing regional traffic flows.MoveBW is overseen by a consortium of six companies, led by Robert Bosch GmbH: transportation solutions company highQ, parking-space operator Parkraumgesellschaft Baden-Württemberg, TraffiCon GmbH, PRISMA Solutions GmbH, and MRK Management Consultants. The moveBW project began in mid-2016 and will end in late 2017.

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We have this platform built to all those in the areas of "Digital School" and "Digital Media" are interested in a central, national point of contact to offer, on which it is to exchange and cooperate can. In addition, we would like to inform you about the use of IT in the Cologne educational landscape.

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HamburgService - Kita-Infosystem

Solo self-employed persons are understood to be persons who carry out an independent activity on their own, ie without salaried employees. In the creative industry, there is an above-average proportion of solo self-employed compared to other sectors of the economy. People who offer creative services or products without being hired are faced with particular challenges in practice because they have to deal intensively and permanently with questions of their own positioning, customer acquisition, marketing, target groups, etc. Many of our offerings are tailored to the needs of solo freelancers in the creative industry. 

As an incubator, since 2013 we have been promoting innovative startups from the higher education sector. Our seat is in the Harburg inland port. Our origin lies at the Technical University of Hamburg. Within the scope of the funding program »EXIST-Founding Culture - The Founders' College« we were supported by the Federal Ministry of Economics and Technology (BMWi) for more than five years. Currently we are part of the "beyourpilot" project of the Department of Economy, Transport and Innovation (BWVI).

The hot. Hamburger ExistenzgründungsInitiative is the first point of contact for anyone looking for self-employment in Hamburg. All the threads of Hamburg's most important start-up initiatives come together here.

The northern German city intends to increase the quality of top-down initiatives, boosting economic growth and reducing the burden of bureaucracy. Within the course of the project, Hamburg will gather citizen input on such topics as the most popular locations for new playgrounds, as well as the most desirable positions for the planting of new trees in public zones. Citizen’s choice for a tree or playground location made in a map should be automatically supported by the systems feedback function, where the citizen will get information about his or her choice based on the data provided by the system. The data (all available as open data on the transparency portal) for the feedback are, for example, noise mapping, solar potential mapping, current tree population, buildings, legally binding land-use plan, and cadastral parcels for the tree as well as green space, land-use zoning, existing playground locations, public transport network and stations, administrative units, inhabitants per unit for the playground location.

Open Data Hamburg The data previously published under the Open Data Portal can now be found here in the Transparency Portal.

Berlin: Invest in a city with a bright future Do you plan to invest in Berlin, start a company here, or relocate your headquarters here? Smart move! Your company can also benefit from the excellent local conditions in the German capital. The Berlin economic development corporation, Berlin Partner for Business and Technology, will support you while your company transfers to the new location, providing help with enterprise development and the transfer of technology with tailored service packages. Berlin Partner's experts can provide you with comprehensive and free advice about Berlin at the Business Location Center.

The waste management strategy for Land Berlin also addresses the high-quality recycling of building materials. Every year, about 1 million tonnes of recycling concrete is generated in Berlin. An on-going investigation of the environmental and climate impacts of mate­rial flows in Berlin has shown, among other things, that the utilisation of recycled con­crete in the building industry could make an important contribution to increasing resource efficiency.

The waste management strategy adopted by the Berlin House of Representatives for the period 2010 to 2020 also provides for an extension of the annual waste audits to provide a comprehensive report on material flows, and climate and environmental impacts for non­hazardous waste, in order to improve the control and evaluation of waste material flows. Therefore the Senate Environment Department, partially financed through the “Climate Protection – Sector strategies” programme of the Federal Ministry of the Environment, commissioned the IFEU Institute Heidelberg and the ICU Berlin to develop a plan for the im­plementation of exemplary, climate-friendly waste management measures for Land Berlin.

The public acquisition system can play an important role in a modern closed-cycle econo­my. Every year, official bodies in the city, from the city and district administrations to the public corporations, statutory bodies and public-law foundations purchase products and services costing some EUR 4 to 5 billion. When placing orders, a considerable contribution can be made to environmental protection by giving preference to environmentally-friendly products and materials and to processes which reduce the impact on the environment. In this way it is not only possible to conserve resources such as energy and water, but also to prevent threats to health and the environment.

You are looking for a family doctor in Reinickendorf, who specializes in diabetes? Or do you want to find out about the consultation hours of your dermatologist? In our medical directory, you will find almost all outpatient doctors in Berlin - including information on qualifications. Also use the advanced search with even more keywords. The information is based on the Kassenärztliche Vereinigung Berlin by the doctors and psychotherapists themselves announced consultation hours.   You can also have office hours displayed on weekends and / or holidays that differ from regular office hours. Days on which special hours are offered appear with a selection box at the bottom of this page

Wheelmap.org is an online map for wheelchair accessible places. Everyone can easily find, register and change places on the website or via an iPhone - as with Wikipedia. The platform went online in September 2010. Already after half a year, volunteers have registered over 40,000 places, every day 100 new places are added. Since November 2010, there is also the free iPhone app.A simple traffic light system marks the wheelchair accessibility of the places: Green means unrestricted access. For example, orange marked places do not have a toilet. Locations that are displayed in red can not be entered by wheelchair users. With the help of this traffic light, people with reduced mobility can find suitable places in their environment and even worldwide. Since places are also listed that are not wheelchair accessible, owners of cafés or other public places are made aware of the problem and encouraged to think about wheelchair accessibility in their rooms.

BürgerBautStadt makes it easier for citizens to participate in construction projects and planning approval procedures. For this purpose, the authors have collected data from various sources and made available on the website as a map, list or e-mail notification. BürgerBautStadt was launched at the ideas competition Stadt Land <Code> of the Open Knowledge Foundation Germany and was supported until May 2013 with a scholarship in the implementation.

Thanks to the free environment zone Android app, you can now check where the environmental zone is located. Whether visiting a foreign city or at home - with the Umweltzone App you know exactly which roads you can drive. In addition, you can read what environmental badge you need. The application informs about adopted changes to the course of the environmental zone and about approved plaques, so that you know in time. For background information on topics such as pollutant groups, particulate matter, health, badges for motorcycles and cars, penalties and exemptions, you can check the FAQs. Last but not least, we refer to some websites that deal extensively with the subject of the environmental zone.

This application for Android devices displays the locations of waste glass containers on a map. Currently only data from the district Charlottenburg-Wilmersdorf is published - as soon as further locations are published, they can be applied to the application. The application is free, the source code is freely available.

The tourism app CultiMapp opens up new possibilities to explore Berlin by linking and enriching the Berlin 3D city model with cultural information. The integrated 3D viewer allows you to fly over interesting buildings near your own location. In the future, historical photographs and detailed shots will also be integrated into the viewer to make Berlin's cultural heritage visible.

naturtrip.org is the first public transport information where you do not have to know the destination. After all, when it comes to excursions, one usually does not know exactly where one wants to go, but only what one plans to do. Namely eat delicious, relax in the spa, dozing or paddling in the sun. Therefore one searches with naturtrip.org from A for "Strandbad" or "Kanuverleih". And how long you want to be on the road. 30 minutes, 60 minutes or more. Then you get on the map exactly the destinations shown, which can be reached from your own location in 30 min or 60 min currently by train, bus or bike. 

How often are streets cleaned in Berlin? With the interactive map application street cleaning in Berlin, the cleaning frequency per week can be called up for each street in the Berlin city area. Additional information can be retrieved simply by clicking on the respective street - What is being cleaned? - Who is responsible and how high are the fees for the residents. The app can be used in any web browser on any device. The application is based on the geodata from the street cleaning directory of the Berliner Stadtreinigung (BSR) and is operated via the mapping platform ArcGIS Online .

Little project that aims at raising awareness for accessibility in public transport systems. It uses a simple slider to visualize how the public transport system looks like if all the non-accessible stations are erased. There is an elaborate how-to available, suitable for non-coders on the projects github site.

This campaign helps people experience what multimodal mobility is like when you don't have your own car. It is aimed at car owners who actually only need their car sporadically. During a four-week trial period, testers were able to try out a wide range of sponsored mobility solutions, all with the goal of making the transition to multimodal mobility easier. Focus Areas The project builds on the initial results of the New Mobility Berlin project which discovered that car owners will only switch to multimodal transportation and give up their own cars if they can first give it a trial run. In order to facilitate the switch, they need to experience the existing multimodal mobility offerings in Berlin. These offerings also need to be expanded locally as mobility-as-a-service (MaaS) adapted to the needs of users.  A sponsored mobility package of Berlin public transport and the city's car sharing companies made it possible for car owners to try out living without a private car. The test also provided important information about user requirements for MaaS.

Data Scientists Build Pothole Dashboard

London Parking

A web portal and mobile application launched in January 2016, that is slated to be Singapore’s first one-stop online health information and services portal. Functions as the digital healthcare companion for every citizen by equipping citizens with the information, knowledge, tools and services to help them take greater ownership of their own health and wellness. A milestone project under Ministry of Health’s (MOH) Health IT Masterplan (HITMAP), healthcare institutions are now also connected with one another to provide continuity of care for patients.

Medifund is an endowment fund set up by the Government to help needy Singaporeans. Medifund is a safety net for patients who face financial difficulties with their remaining bills after receiving Government subsidies and drawing on other means of payments including MediShield Life, private Integrated Shield Plans (IPs), Medisave and cash. Set up in April 1993 with an initial capital of $200 million, the Government will inject capital sum into the fund from time to time, e.g. when budget surpluses are available. The interest income generated from the capital sum is used to provide financial assistance for healthcare bills.

Medisave is a national medical savings scheme which helps individuals put aside part of their income into their Medisave Accounts to meet their future personal or immediate family's hospitalization, day surgery and certain outpatient expenses.

WELCOME TO HEALTHHUB

ActiveSG is an all-encompassing and inclusive national movement for sport, brought to you by Sport Singapore. Poised to be a lifestyle destination for Singaporeans, ActiveSG will offer individuals, families and communities ample opportunities to experience and share the joy of living better through sport. Come join our national movement for sports and get active with a diverse and exciting line-up of sporting activities suited for all! Our sport facilities which are conveniently located all over Singapore are open to all! You can also sign up for ActiveSG membership registration to enjoy further privileges. Membership registration is free for all Singaporeans and Singapore Permanent Residents!

We digitise, connect and analyse Singapore’s health ecosystem.  Our ultimate aim is to improve our population’s health and health administration by integrating intelligent, highly resilient and cost effective technologies with process and people.

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UBE2A

UBE2A

What they would like would be to have additional powers conferred upon them, rather than to have existing ones contrated. Perhaps the system now everywhere in use in Upper Canada would be beneficial in the Townships.

Local works naturally fell within the scope of local governments, and would undoubtedly be under the immediate influence of the municipal councils, but all the works of a really public character would be under the General Legislature; such, he meant, as were connected with the general policy of the whole country.

The Municipal institutions of the country must necessarily come under the care of the local Legislatures, and in fact the local Legislatures were themselves municipalities of of a larger growth. They were charged with the administration of local affairs, and must be allowed to delegate such powers as they thought might be safely entrusted to the smaller divisions of the country as laid out into townships and parishes.

(BB/N023129/1)

It is at that age of aptness, docility & emulation of the practices of manhood, that such things are soonest learnt, and longest remembered.

To enlighten them with mathematical and physical sciences which advance the arts & administer to the health, the subsistence & comforts of human life

To instruct the mass of our citizens in these their rights, interests and duties, as men and citizens,

But in this point of View the Anglo-Saxon is of peculiar value. We have placed it among the modern languages because it is in fact that which we speak, in the earliest form in which we have knowledge of it. It has been undergoing, with time, those gradual changes which all languages, antient and modern, have experienced: and even now, needs only to be printed in the Modern character and Orthography, to be intelligible in a considerable degree to an English reader.

To instruct the mass of our citizens in these their rights

Spanish is highly interesting to us, as the language spoken by so great a portion of the inhabitants of our Continents, with whom we shall possibly have great intercourse ere long

It was the degree of centrality to the white population of the state which alone then constituted the important point of comparison between these places: and the board, after full enquiry & impartial & mature consideration, are of opinion that the central point of the white population of the state is nearer to the central college, than to either Lexington or Staunton by great & important differences, and all other circumstances of the place in general being favorable to it as a position for an University, they do report the central college in Albemarle to be a convenient & proper part of the State for the University of Virginia.

It will form the first link in the Chain of an historical review of our language through all its successive changes to the present day, will constitute the foundation of that critical instruction in it, which ought to be found in a Seminary of general learning

To enlighten them with mathematical and physical sciences which advance the arts & administer to the health, the subsistence & comforts of human life:

They will be more advanced than we are, in science and in useful arts, and will know best what will suit the circumstances of their day.

Medicine, when fully taught, is usually subdivided into several professorships, but this cannot well be without the accessory of an hospital, where the student can have the benefit of attending clinical lectures & of assisting at operations of surgery. With this accessory, the seat of our university is not yet prepared, either by its population, or by the numbers of poor, who would leave their own houses, and accept of the charities of an hospital.

To develope the reasoning faculties of our youth, enlarge their minds cultivate their morals, & instil into them the precepts of virtue & order

that to secure Ourselves where we are, we must tread with awfull reverence in the footsteps of Our fathers

This doctrine is the genuine fruit of the alliance between church and State,

The tender age at which this part of education commences, generaly about the tenth year, would weigh heavily with parents in sending their sons to a school so distant as the Central establishment would be from most of them

Daily walks, jogs, or play sessions can pay huge dividends when it comes to your dog’s health. Not only will exercise help keep your dog fit and trim, regular activities can help channel your dog’s energy into a positive directio

Some key themes arise from the two NNG reports on iPad usability: App designers should ensure perceived affordances / discoverability There is a lack of consistency between apps, lots of ‘wacky’ interaction methods. Designers should draw upon existing conventions (either OS or web) or users won’t know what to do. These are practical interaction design observations, but from a particular perspective, that of perceptual psychology. These conclusions are arrived at through a linear, rather than lateral process. By giving weight to building upon existing convention, because they are familiar to the user, there is a danger that genuinely new ideas (and the kind of ambition called for by Victor Bret) within tablet design will be suppressed. Kay’s vision of the Dynabook came from lateral thinking, and thinking about how children learn. Shouldn’t the items that we design for this device be generated in the same way?

A first list of projects are available here but more can be found by interacting with mentors from the Pharo community. Join dedicated channels, #gsoc-students for general interactions with students on Pharo slack. In order to get an invitation for pharoproject.slack.com visit the here Discuss with mentors about the complexity and skills required for the different projects. Please help fix bugs, open relevant issues, suggest changes, additional features, help build a roadmap, and interact with mentors on mailing list and/or slack to get a better insight into projects. Better the contributions, Better are the chances of selection. Before applying: Knowledge about OOP Basic idea about Pharo & Smalltalk syntax and ongoing projects Past experience with Pharo & Smalltalk Interaction with organisation You can start with the Pharo MOOC: http://files.pharo.org/mooc/

and to correct solitary people

as he faced the firing squad

That’s my fear. That I’m going to get through all this and be like, “I don’t know.”

She is not a small talker or a beater around of bushes. You discuss whatever it is you came to discuss full-on, looking it squarely in the face. She has no time for bullshit.

WHAT DOES IT MATTER WHO IS SPEAKING/' SOMEONE SAID, "WHAT DOES IT MATTER WHO IS SPEAKING": Beckett, Foucault, Barthes Alastair Hir

overworked and exhausted

mental state