Test for thialysine resistance

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

For determiningthe intracellular pH from fluorescence intensity values of CFDA-SE-loaded cells, anin vivocalibration curve was prepared between fluorescent intensity and pre-adjusted environmental pH values. Briefly,CFDA-SE-loaded wild-typeC. glabratacellswere incubatedwith 0.5 mM carbonyl cyanide m-chlorophenylhydrazone (CCCP; Sigma# C2759) at 30 ̊C for 10 min in 50 mM CP buffer adjusted to different pH values ranging from 4.0 to 7.5, with an interval of 0.5 unit.CCCP is an ionophore which dissipates the plasma membrane pH gradient, thus, rendering the intracellular pH similar to the extracellular pH. Fluorescent intensities were determinedand a calibration curvewas plotted between the ratio of intensity at 490 to430 nm versuspH.A polynomial distribution of fluorescent intensity signal and pH was observed for CFDA-SE probe(Figure2.1)and the graphequation was used todeterminethe intracellular pHof C. glabratacells

In vivointracellular pH calibration curve

and colony purified on CAA plate. 15% glycerol stocks were made for two independent transformants and stored at -80 ̊C

C. glabrataCgYPS7ORFwas cloned in a self-replicating pGRB2.2plasmidwhich contains C. glabrata CEN-ARS, S. cerevisiaeURA3gene, S. cerevisiaePGK1promoter and C. glabrataHIS3-3′ untranslated region. For cloning CgYPS7in pGRB2.2,CgYPS7ORF (1.764 kb) was PCR-amplified from the wild-type genomic DNA with high fidelity Platinum PfxDNA polymeraseusing primers carrying restriction sites for XbaIand XhoI. The1.764 kb amplifiedPCR product waspurified with QIAquick PCR purification kit (Qiagen # 28104),digested with XbaI and XhoI and cloned in the pGRB2.2plasmid at XbaI–XhoI sites in the multiple cloning site (MCS)region downstream of the PGK1promoter.Positiveclones were verified by PCR, sequencing and complementation analysesofCgyps7∆mutant. Yeast transformantsobtained by lithiumacetate methodwere selected on plates lacking uracil

Cloning of CgYPS7gene

All experiments in this studywere performed with log-phase cellsunless otherwise mentioned. For obtaining log-phase cells, overnight YNB-or YPD medium-grown yeast cellswerere-inoculated in fresh YNB or YPD medium to an initial OD600of 0.1-0.2.Cells were incubated at 30 ̊C with shaking at 200 rpmtill the OD600reached to 0.4-0.6 OD. After incubation, log-phase cellswere collected bycentrifugation at 4,000 rpm for 3 min,washed once with the same medium and usedforfurtheranalysis

Cultivation of logarithmic-phase cell culture

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

was washed three times with PBS to remove non-adherantC. glabratacellsand Lec-2 cells were lysed in 5% SDS. Lysates were transferred totubes containing scintillation fluidand radioactive counts obtained were considered as ‘output values’. Percentage adherence wasdetermined using following formula

Adherence of C. glabratacells toLec-2 epithelial cells wasmeasured as described previously(Cormack et al., 1999).Lec2cells were seeded ina 24-well tissue culture plate at a seeding density of 5X105cells per well and allowed to adhere for 12 h. After 12 h,medium supernatant was discarded by inverting the plate in a reservoir and cells were washed thrice with PBS. Lec2 cells were fixed in 3.7% para-formaldehyde for 15 minfollowed by 2 PBS washes. PBS containing antibiotics, penicillin and streptomycin,was added toeach well of the 24-well plate and Lec-2 cellswere stored at 4°C.For adherence measurement,strains were taken out either on YPD or CAA mediumandgrown at 30°C for 2 days. Single colony of a C. glabratastrain wasinoculated in 10 ml CAA medium ina 100 ml culture flaskand allowed to grow at 30°C for 16-20 h. 100 μlyeast culture wasreinoculated in fresh 5 ml CAA liquid medium in a 15 ml polypropylene tube. 200 μCi of S35(Met:Cys-65:25) INVIVO PROTWIN labelmix(JONAKI, India) was added to thetube and cultures were grown at 30°C for 16-20 h for radiolabeling of C. glabratacells. C. glabratacells from 1 ml culture were harvested and washed threetimes with PBS to remove residual S35(Met:Cys-65:25) labeling mix from medium supernatant. Next,cells were resuspended in 1 ml PBS. OD600was measured and cell suspensions of 0.5 OD600were prepared. PBS was aspirated out of the wells of 24-well plate containing fixed Lec-2 cells. 200 μl of S35(Met:Cys-65:25)-labeled C. glabratacell suspensions were added to each well. To determine the total amount of radioactivity present in labeled C. glabratacell suspension, 200 μl of S35(Met:Cys-65:25)-labeledC. glabratacell suspensions were transferred to a scintillation vial containing scintillation fluid. Radioactive counts present in this fraction were considered as ‘input values’. For measurement of yeast adherence to Lec-2 cells, plates were centrifuged at 1,000g for 5 min and incubated for 30 min at room temperature. Following incubation, each wel

Adherence assay

After 2-3 h exposure,phosphorimagerscreenwas scannedon Fuji FLA-9000 to acquire hybridization images. Next,signal intensity for each spot on the membrane for both input and outputsampleswas quantifiedusing Fuji Multi Gauge V3.0 software andpercentage intensity foreach spot relative tothe whole signal intensity ofthe membranewas determined.To identify mutants with altered survival profiles,ratio of output (Op) to input (Ip) signal for each spot (oligonucleotide tag)present on the membranewas calculated.Mutantsdisplaying at least 6-fold higher and 10-fold lower survival were selectedas “up’ (Op/Ip= 6.0) and ‘down’ (Op/Ip = 0.1) mutants, respectively

Data analysis

Single colony of C. glabratastrains wasinoculated in 10ml YPD-liquid medium and grown at 30°C with constant shaking at 200 rpm for 14-16 h. Overnight culture was used to inoculate 10 ml YPD broth to an initial OD600of 0.1 and culture was grown for 4-5 h to obtain log-phase culture. Log-phase C. glabratacells were harvested in 15 ml sterile polypropylene tubesby centrifugation at 4,000 rpm for 5 min. Harvested cells were washed with10ml sterile water,resuspendedin 1 ml sterile water and transferred to a 1.5 ml microcentrifuge tube. Cells were harvested at 4,000 rpm for 5 minand resuspended in 100 μl of100mM lithium acetate solution.Yeast transformation cocktail was prepared in a 1.5 ml microcentrifuge tube by mixing 240 μlpolyethylene glycol(50%), 36μl lithium acetate(1 M) and25μlheat-denatured single stranded carrier DNA(2 mg/ml). 50 μlC. glabratacell suspension and 50 μltransforming DNAwas added to the transformation cocktail, mixed well andincubatedat 30 ̊C for45 min. 43 μlDMSO was added and cells were subjected to heat shock at 42 ̊Cfor 15 min. After the heat shock, cells were transferred to ice for 10-15 seconds, centrifuged at 4,000 rpm for 5 min and supernatantwas removed.Cells wereresuspended in 200 μlsterile water andspread platedonappropriate selectionmedium. Plates wereincubatedat30 ̊Cfor 2-3 days

Yeast transformation

Forinfection of THP-1 cells with single C. glabratastrain, PMA-treatedTHP-1 monocytes were seeded in 24 wellcell culture plate toa seeding density of 1 million cells per well. To prepare C. glabratacells for macrophage infection, single colony of the desiredstrain wasinoculated in YPD medium and allowed to grow for 14-16 hat 30°C. C. glabratacellsfrom 1ml overnight culture were harvested, washed with PBS andcell density was adjustedto 2X107cells/ml.50 μl of thisC. glabratacell suspension wasinfectedto macrophages to a MOIof 10:1. Two hours post infection, infected THP-1 macrophages were washed thrice with PBS to removenon-phagocytosed yeast cells and medium was replacedwith fresh prewarmed medium. Atdifferent time points post infection,infected THP-1 macrophages were washed with PBS three timesandlysed in 1 mlsterilewater. Lysates were collected by scrapping the wells with a micropipette tip, diluted in PBS and appropriatelysatedilutions were platedon YPD agar medium. Plates wereincubated at 30°C and colony forming units (CFU) were counted after 1-2 days. Final CFUs per ml were determined by

multiplying CFUs with dilution factor and fold-replication was determined by dividing the CFUs obtained at 24 h time-point by 2 h CFUs

Single infection assay

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

Of theligation mixture,2μl (of total volume of 10 μl reaction)was added to atube of 100μlultra competent DH5α bacterial cells and incubated in ice for 30 minutes. The tubewas quickly transferred to a water bath maintained at 42°C to give a heat shock for 90 seconds and again quickly transferred to ice. 1ml of LB broth was added to the tube and then incubated at 37°C for 1 hour. The bacterial cells were then pelletdownby centrifugation at 6000 rpm for 5 minutes and plated on LB agarcontaining appropriate antibiotic

Transformation of ligated DNA

Automated DNA sequencing on plasmid templates or on PCR products was carried out with dye terminator cycle sequencing kits from Perkin-Elmer on an automated sequencer (model 377, Applied Biosystems), following the manufacturer’s instructions

DNA sequencing

BCA (Bicinchoninic acid) method was used to determine the proteinconcentrationin various samples. The Cu2+ions from cupric sulphate (present inBCA reagent B) reagent arereduced to Cu+by the protein in an alkaline medium. The cuprous ion (Cu+) then combines with BCA (present in BCA reagent A) to give a purple colour whose intensity is proportional to the amount of protein present in the samples. This intensity is measuredby colorimetry at 562 nm. BCA reagent was prepared by mixing reagent A with reagent B in avolumeratio of 50:1. A standard curve was generated using increasing concentrations of BSA (2-10μg) in a 25μl reaction, in a 96 well plate. Cell lysates were also dilutedto same volume in parallel wells. 200μl of BCA reagent was then added to each well and incubated at 370C for 30 minutes. The absorbance readings were then takenin a spectrophotometer at 562 nm. Total protein was quantified by calculation of the slopes of regression lines ofabsorbanceand BSA standards

Protein estimation

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

The method described earlier by Gilliesand co-workerswas slightly modified and followed (Gillies et al.,1986). Briefly, parentaland profilin-stable cells were seeded in triplicates at a density of 20,000 cells per well of a 24-well culture plates. Each day after seeding, cells were washed with PBS and stained with 0.2% crystal violet in 2% ethanol for 15 minutes. Vigorous washing was done with PBS to remove excess dye. Crystal violet dye was then eluted using 1% SDS solution with extensive pipetting and diluted 10 fold. Absorbance of the extracted dye was then determined at 570 nm in a spectrophotometer. Absorbance data based on triplicate set of samples for each experimental condition were then averaged for each time point to generate a growth curve

Cell proliferation assay

6X DNA loading dye

Agarose gel

TAE

For DNA electrophoresis

(e) Stacking polyacrylamide gel

growntill the OD600reached 0.8-1.0. Cells equivalent to 1OD600of each culture was taken for the labellingof total protein. Cells were washed in methionine-free synthetic complete medium(SC-Met) twice, suspended in SC-Met mediumcontaining 25μCi/mLof 35S Met-Cys twin label mixand incubatedfor 1min, 5 min and 15min. Cells were washed twice in ice-cold SC-Met medium twice and suspended in 500 μL of Tris-salinecontaining protease inhibitor cocktail.To this,300 μL ofglass beads (0.45-0.6mm diameter)were added and cells were lysed for 10 min by bead beating (with intervals of 1 min on time and 30 sec off time).The lysate was centrifuged at high speed for 15 minat 4°C. To the supernatant,sodium deoxycholate was added to a final concentration of 0.1 mg/mLand incubated on ice for 30 min. To this solution, 20% trichloroacetic acid was addedto a final concentration of 6%, incubated for 1 h on ice, and centrifugedat high speedfor 20minat 4°C. The pellet was suspended in 300 μL of Tris-saline and counted inaliquid scintillation counter (Perkin Elmer-Tricarb 2900). The cpm values obtained were plotted using GraphPad Prism5

Wild type and knock out yeast strains were grown in YPD (Difco) whereas synthetic complete medium without uracil was used for the KCS1 complementedstrains. Overnight grown yeast were subcultured in appropriate medium at 0.2 OD600and

Protein synthesis analysis

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

The plasmid-DNA/PEI mixture was incubated for 15 minutesat room temperature.The mixture was added to cells,andmixed properlyby rocking the culture plate back and forth. Cells were incubated at 37°C in a CO2 incubator.The transfected cells were harvested at 24-48 hours post-transfection

Cells were plated inthe cell culture dishes one day before transfection in RPMI1640 supplemented with FBS and penstrep (complete medium). All the reagents were brought to room temperature before starting transfection. Plasmid-DNA was diluted in serum-free medium and PEI was added(Table 9)Table 9: PEI plasmid-transfection methodology

Plasmid transfection using PEI

Table 8: Lipofectamine plasmid-transfection methodology

For transfection with Lipofectamine, cells were plated in antibiotic-free medium 24 h before transfection and were transfected at a confluency of 70-80% as per the manufacturer’s protocol. The plasmid of interest was incubated in serum free media,and Lipofectaminewas incubated in serum free media forseparately5minutes. The plasmid and the Lipofectamine mixtures(Table 8)were mixedgentlyand incubated at room temperature for 20 min.;thetransfection mixture was added dropwise to the cells. Transfection media was replaced with the fresh complete medium after 6 hrs.of transfection and cell are harvested after 24 hours

Plasmid transfection using Lipofectamine 2000

293T cells or HeLa cells were transfected with various plasmids as per the designed experiments using Lipofectamine 2000 (Invitrogen) reagent or PEI

PlasmidTransfectionof mammalian cells

To study the virulence of Xanthomonas oryzaepv. oryzicolastrains on rice plant two different inoculation methods, syringe infiltration and wound inoculation methods, were implimented. For infiltration method, bacterial suspension comprising of 1 × 108 cells/ml were infiltrated with needleless syringe into leaves of 4 to 6 week-old rice cultivar of susceptible Taichung Native-1 (TN-1) (Hopkins et al., 1992; Wang et al., 2007). Wound inoculation method was carried out by dropping an aliquot of 20 μl bacterial suspension comprised of 1 ×108cells/ml onto fully expanded leaf of 6-8 week green-house grown Taichung Native-1 cultivar of rice, and pricking with sterile needle for facilitating the entry of Xocinside the leaves throgh wound. For inititation of disease symptom, the inoculated plants were incubated in greenhouse with minimum and maximum temperatures of approximately 25 to 30 °C, respectively, and a relative humidity of approximately 60%. Water soaking symptom and lesion development was measured 4 to 10 days after inoculation. Likewise, for infiltration by wound inoculation method, lesion length was measured 14 days after inoculation. In both the cases, no lesions were observed in control experiments in which the leaves were inoculated with sterile wate

Virulence assay on rice plant

BXOR1, ΔrpfFand ΔrpfF(pSC9) strains were grown to OD600of 1 in rich media (PS), PS + 50 μM 2,2’-dipyridyl (DP) and PS + DP + 30 μM FeSO4. RNA was isolated by Trizol (Invitrogen) method as described above. Optimal primer and cDNA concentrations were standardized, and qRT-PCR was performed using ABI 7500 Fast Real-Time PCR system (Applied Biosystems). In brief, 1 μl cDNA, 0.25 picomoles of gene specific primers and 10 μl 2X SYBR GREEN qPCR Mastermix (Qiagen)were mixed in the wells of 96-well PCR plate (Axygen). Final reaction volume was adjusted to 20 μl with nuclease-free water. Transcript levels were quantified with an end-point value known as Ct(cycle thresold) value. Expression of 16S rRNA was used as an internal control. The Ct values defines the number of PCR cycles required for the fluorescent signal of SYBR green dye to cross beyond the background level. Fold-change in transcript expression was determined using following formula.Fold change in expression = 2-ΔΔCtΔΔCt= ΔCt treated-ΔCt untreatedΔCttreated = Ctvalue for the gene of interest under treated condition -Ct value for the internal control gene (16S rRNA) under treated conditionΔ Ctuntreated = Ct value for thegene of interest under untreated condition -Ct value for the internal control (16S rRNA) gene under untreated condition

Primers for real-time PCR analysis were designed using Primer3 plus software and are listed in Table 2.2.For RNA isolation, X. oryzaepv.oryzaewild-type, rpfFmutant, rpfF/CG8 complemented strains were grown in PS medium at 28°C for 28 h at 200 rpm. Similarly, for RNA isolation from X. oryzaepv. oryzicola, the Wild-type

Quantitative real-time PCR

E.coliDH5α strain was transformed with plasmids carrying appropriate inserts to generate clones, and Xanthomonas deletion strains. Ultracompetent cells stored at -80°C were thawed on ice for 5-10 min. 5 μl ligated plasmid was added to 100 μl ultracompetent cells and incubated on ice for 30 min. Next, competent cells were subjected to heat shock at 42°C for 90 seconds. Cells were immediately transferred on ice for 2-3 min. Next, 1 ml LB medium was added and cells were allowed to recover for 1 h on a shaker incubator set at 37°C. After the recovery, cells were centrifuged at 3000 g for 3 min. Medium supernatant was discarded and cells were resuspended in 100 μl fresh sterile medium. Cells were plated on LB agar containing appropriate antibiotics. Plates were incubated at 37°C for 12-16 h

E.colitransformation

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

For bacterial isolates, a single colony from a nutrient agar slant was inoculated into 50 ml of nutrient broth in a 250 ml Erlenmeyer flask. These flasks were incubated at 37±1°C in a incubator shaker till an optical density of 0.6 at 660nm. Now these cultures were used to inoculate 50 ml of the tannase production medium in 250 ml Erlenmeyer flasks using 2% v/v inoculum. These flasks were incubated at 37±1°C in an incubator shaker (Multitron AG-27; Switzerland) at 200 rpm for 72h. The experiments were carried out in triplicates. Samples (2.0 ml for bacteria and same for fungi) were withdrawn at regular intervals of 12h upto 72 h. The samples thus obtained were centrifuged at 10,000 rpm in a refrigerated centrifuge (SIGMA 4K15 Germany) for 10 min at 4°C. The supernatant/s were analyzed for tannase activity

For fungal cultures, spores were harvested from 72 hour old cultures grown on PDA/Tannic acid agar slants by adding 10 ml of sterilized normal saline and a few drops of sterilized Tween-80 followed by vortexing. The spore suspension was filtered through sterile cotton filter to ensure that mycelial filaments are removed. The spores were counted using a haemocytometer (Neubaeur). Approximately, 5X106 spores were inoculated in 50 ml of tannase production medium in 250 ml Erlenmeyer flasks. These flasks were then incubated at 30±1 and 37±1°C in an incubator shaker (model G25KC, New Brunswick Scientific, NJ, USA) at 200 rpm

Quantitative assay

spectro-photometrically at 340 nm. For wild-type cells,mitochondrial aconitae activity was normalized to 100 % and for mutants the relative aconitase activity percentages were calculated

To determine aconitase activity, mitochondria were isolated as described by Meisinger et al. Briefly, YPD-grown C. glabratacells (500 OD600) were subjected to spheroplasting followed by homogenization (15 strokes) with glass Teflon homogenizer. To collect mitochondria, homogenate was centrifuged at 13200 g for 20 min in a refrigerated centrifuge set at 4°C. The mitochondrial pellet was resuspended in SEM buffer (250 mM sucrose, 1 mM EDTA, 10 mM Mops-KOH, pH 7.2) and stored at -80°C until further use. Mitochondrial aconitase activity was estimated by using method as described by Bulteau et al. Mitochondrial protein samples (5 μg) were prepared in KH2PO4buffer (25 mM, pH 7.2) containing 0.05 % Triton X-100. The samples were incubated with sodium citrate (1 mM), MnCl2(0.6 mM), NADP (0.2 mM) and isocitrate dehydrogenase (1 U/ml) for 20 min at room temperature. Isocitrate dehydrogenase catalysed reduction of NADP was recorded

Measurement of aconitase activity

were chosen for qPCR. For all qPCR reactions,0.4 μl of cDNA template was used in a 20 μl reaction volume. Reactionswere performed anddata wereanalysed in ABI7500 real-time qPCR machine. Amplified products were run on 2% agarose gel to confirm amplification ofthecorrect size product. CTvalues of respective products were normalized with corresponding CTvalue of the housekeeping gene CgACT1. Relative change in expression was determined by comparative CTmethod,also referred as 2-∆∆CTmethod, utilizing following equation.Fold change upon treatment=2-∆∆CT∆∆CT=∆CT Treated-∆CT Untreated∆CTTreated= CTvalue for gene of interest upon treatment-CTvalue of internal control (CgACT1)upon treatment∆CTUntreated= CTvalue for gene of interest without treatment-CTvalue of internal control (CgACT1)without treatmentThe reaction cycling conditions were as follows1)95°C for 10 min (initial activation)2)95°C for 15 sec (denaturation)3)55°C for 30 sec (annealing)4)72°C for40 sec (extension)5)Go to step 2 (40 cycles)6)72°C for 10 min (final extension)

MESA GREEN qPCR mastermix (RT-SY2X-03+WOULR) supplied by Eurogentech was used in all qPCR experiments. Primers for real-time qPCR experiments were designed by using the Primer3 plus software to obtain 120-200 bp amplification products. Standardization of optimaltemplate and primer concentrationconditionswas done in a PCR reaction and concentrations resulting in good amplification withoutprimer dimers

Quantitative Real-time PCR(qPCR)

Estimation of cytokine production by THP-1 macrophages upon infection with C. glabratacells

were chosen for qPCR. For all qPCR reactions,0.4 μl of cDNA template was used in a 20 μl reaction volume. Reactionswere performed anddata wereanalysed in ABI7500 real-time qPCR machine. Amplified products were run on 2% agarose gel to confirm amplification ofthecorrect size product. CTvalues of respective products were normalized with corresponding CTvalue of the housekeeping gene CgACT1. Relative change in expression was determined by comparative CTmethod,also referred as 2-∆∆CTmethod, utilizing following equation.Fold change upon treatment=2-∆∆CT∆∆CT=∆CT Treated-∆CT Untreated∆CTTreated= CTvalue for gene of interest upon treatment-CTvalue of internal control (CgACT1)upon treatment∆CTUntreated= CTvalue for gene of interest without treatment-CTvalue of internal control (CgACT1)without treatmentThe reaction cycling conditions were as follows1)95°C for 10 min (initial activation)2)95°C for 15 sec (denaturation)3)55°C for 30 sec (annealing)4)72°C for40 sec (extension)5)Go to step 2 (40 cycles)6)72°C for 10 min (final extension)

MESA GREEN qPCR mastermix (RT-SY2X-03+WOULR) supplied by Eurogentech was used in all qPCR experiments. Primers for real-time qPCR experiments were designed by using the Primer3 plus software to obtain 120-200 bp amplification products. Standardization of optimaltemplate and primer concentrationconditionswas done in a PCR reaction and concentrations resulting in good amplification withoutprimer dimers

Quantitative Real-time PCR(qPCR)

E. colibacterial strain DH5α was taken out on LB-agar mediumfrom -80°C freezer and incubated at 37°C for 14-16 h. To obtain the starter culture,single bacterial colony was inoculated in 25 ml of SOB medium ina25 ml flask. The flask was incubatedfor 6-8 hat 37°C with continuous shaking at 200 rpm. Next, 2, 4 and 10 ml of the starter culture was inoculated in three different 1 litre flasks each containing 250 ml of SOB medium. Cultures were incubated overnightat 18°C with continuous shaking at 200 rpm. After overnight incubation, OD600 of allthree cultures were monitored after every 45 min interval till OD600of any of the three cultures reached 0.55. These cells werekept onice for 10 min and the other two cultures were discarded.Cells were harvested by centrifugation at 2,500g in a Sorvall GSA rotor for 10 min at 4°C. Supernatant was poured offcompletely andcells were gently resuspended in 80 ml of ice-cold Inoue transformation buffer by swirling the tubes (pipetting was avoided at this step). Followingresuspension, cells were spun down by centrifugation at 2,500g in a Sorvall GSA rotor for 10 min at 4°C and the supernatant was discarded completely. The cell pellet was resuspended gently in 20 ml of ice-cold Inoue transformation buffer by swirling.1.5 ml of DMSO was added to the cell suspension and incubated on ice for 10 min. 50 μl aliquotsof cell suspensionwere dispensed in pre-chilled 1.5 ml microfuge tubes, snap-frozen in liquid nitrogen and stored in -80°C freezer till further use

Preparation of ultra-competent E. colicells

PMA-treated THP-1 cells were seeded toa24-well tissue culture plate to a cell density of 1 million cells per well and allowed to grow for 12 h. After12 hincubation,spent medium was replaced with fresh pre-warmed RPMI-1640 medium andcells were allowed to recover for 12 h before use.C. glabratacells were grown in YNB medium for 14-16 h at 30°C and 200 rpm. 1 ml of theseC. glabratacells were harvested in 1.5 ml centrifuge tubes, washed twice with 1X sterile PBS and the cell density was adjusted to 2x107cells/ml. 50 μl of this cell suspension was used for infection to a MOI of 1:1. Two hours post infection, wells were washed thricewith 1X sterile PBS to remove the non-phagocytosed yeast cells and 1 ml of fresh pre-warmed RPMI-1640 medium was added. Plates were incubated under tissue culture conditions at 37°C and 5% CO2for 24 h. Supernatants were collected in 1.5 ml microfuge tubes, centrifuged at 3,000 rpmto remove the particulate matter, if any, and stored at -20°C until use. Estimation of different cytokines were performed using BD OptEIA ELISA kits as per the supplier’s instructions

Estimation of cytokine production by THP-1 macrophages upon infection with C. glabratacells

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

the same solution and stored overnight at 4ºC. Fixed cells were permeabilized with 100 μL saponin based permeabilisationbuffer and wash buffer for 2 minin the dark. Cells were pelleted down and washed twice with saponin based permeabilisation buffer, centrifuged to pellet the cells and the supernatant was aspirated leaving 30-40 μLbuffer to dislodge the pellet. EdU was detected by adding 350 μLof Click-iT reaction cocktail and incubated for 30 min at room temperature in the dark. Cells were washed once with permeabilisation buffer and DNA content was measured by adding 5 μL Ribonuclease A and 2 μLof cell cycle dye 633 red incubated for 15 min at room temperature in the dark. Cells were analyzed by flow cytometry (FACS ARIA, Becton Dickenson). Data analysis to determine the stages of thecell cycle was performed using FACSDiva (BectonDickenson), and results were plotted using GraphPad Prism 5

Cell cycle analysis by PI staining is based on the DNA content of cells and cannot distinguish G0from G1, and G2 from M phase. One more limitation is that it provides overlapping cell populations in different phases. Alternatively, multi parameter based cell cycle analysis can be performed using EdU (5-ethynyl-2’-deoxyuridine, a BrdU alternative)labeling which exclusively distinguishes cells in S phase from other phases of cell cycle. EdU labeling and cell cycle analysis was conducted using the Click-iT cell proliferation assay kit (C35002, Invitrogen), as per the manufacturer’s instructions. Briefly, cells were grown in 35mm dishes at 30% initial confluence. At 50-60% confluence, cells were treated with 0.2 mM HU for 12 h. After treatment, media containing drug was removed, gently washed twice with PBS and replaced with fresh media. Cells were allowedto recover for different time periods such as 3, 6, 9 and 12 h to observe arrest and release into S-phase. At each time point cells were labeled with 25μM EdUfor 30 min before harvestingby trypsinisation. Harvested cells were washed with 1% BSA in PBS and fixed with 100 μLof ClickiT-fixative containing 3% paraformaldehyde for 15 min at room temperature. After fixation, cells were again washed with 1% BSA in PBS, resuspended in

Cell cycle analysis by EdU labeling

Antibodies

Table 2.3: Antibodies used in this study

The method followed was as described in Miller (1992). Samples for dot-blotting were prepared by mixing 5μg of RNA (in10μl H20) with 30μl of RNA denaturing solution consisting of 1X MOPS, 7% formaldehyde and 50% deionised formamide. The samples were heat denatured at 65oC for 5 min and mixed with equal volume of 20X SSC. The samples wereloaded into the slots of the dot-blot apparatus (Bio-Rad) containing the membrane (pre-soaked in 20X SSC)and gentle suction was appliedusing Millipore vacuum pump. The slots were rinsed twice with 10X SSC. This was followed by the UV-crosslinking, pre-hybridization, hybridization, washing and exposure of the membrane identical to that done in Northern blotting

Dot-blotting

For hybridisation,probe was heated at 95oC for 5 minutes and snap-chilled for 5 minutes and then added to the hybridisation bottles containing the blot. Hybridisation was carried out overnight at 50oC.The probes used and their radioactivity counts (in parentheses) were 5s RNA probe (2.5×106cpm), U73 probe (5×106cpm) and lacZ probes (106 cpm)

Hybridisation of the membrane

DNA sequencing

Automated DNA sequencing on plasmid templates or on PCR products was carried out with dye terminator cycle sequencing kits on an automated sequencer following the manufacturer's instructions byan outsourced sequencing facility

Typically 400-500ng of DNA was used in each ligation reaction. The ratio of vectorto insert was maintained between 1:3 and 1:5 for cohesive end ligation. The reaction was generally performed in 15μl volume containing ligation buffer (provided by the manufacturer) and 0.075 Weiss unit of T4 DNA ligase at 16ºC overnight (14-16 hours)

Ligation of DNA

or absence of a metabolite or a particular temperature. An EOP of ≤0.01 suggests lethality of the strain on the test medium. For strains carrying IPTG-dependent plasmids, EOP was determined by growing the strains overnight in medium containing IPTG and appropriate antibiotic,and spottingserial dilutions (100or 10–1to 10–6) on +IPTG (permissive) and –IPTG (test) plates to observe growth. Theviability is scored by takingratio of the colony forming units per ml (cfu/ml = No. of colonies × dilution factor × 1000/volume of culture spotted (in μl) obtained on the –IPTG plate to that on the +IPTG plate and determinesthe EOP. Likewise, strains carrying Ts plasmids were cultured overnight at 30°C with the appropriate antibioticand the serial dilutions of this culture were spottedat two temperatures 30°C (permissive) and 42°C (non-permissive or test). The ratio of cfu/mlobtained on the test temperature to that on the permissive temperature determined the efficiency of plating at the test temperature

Efficiency of plating (EOP) is a measure of the ratio of number of colonies (obtained from a given volume of a suitable culture dilution) on a test medium to those on a control or permissive medium, and is a measure of cell viability on the former. It is a very sensitive test and is often used for determining the viability of a strain in the presence

Efficiency of plating (EOP)

EDTA 2 mMThis was prepared at 50X concentration and used at 1X concentration. Both TBE and TAE were used as standard electrophoresis buffers.Gel loading buffer with dye Tris-Cl (pH 7.5) 250 mM Bromophenol blue/ Xylene cyanol 0.02% Glycerol 20%INOUE (PIPES) Buffer PIPES (Free acid) 10 mM CaCl2.2H2O 15 mM KCl 250 mM MnCl2.4H2O 55 mM pHwas adjusted to 6.7 with 1N KOH. PIPES gets into solution when the pH is greater than 6.7. MnCl2 was dissolved separately and added with stirring. The pH was then adjusted to 6.7 and solution wasfilter sterilized and stored at –20ºC.Z Buffer (for β-galactosidase assay) Na2HPO416.1 g NaH2PO45.5 g KCl 0.75 gMgSO4.7H2O 0.246 g H2O to 1000 ml pH was adjusted to 7.0 and stored at 4ºC.Pre-Hybridization Buffer 20X Saline-sodium citrate (SSC)3ml50% dextran sulphate2ml50X Denhardt’s solution1m

20%SDS250 μl10 mg/ml Salmon sperm DNA100 μlDEPC waterto 10mlHybridization Buffer Same as pre-hybridisation buffer but contains the radio-labelled probe.SDS sampleBuffer(1X)Tris-HCl, pH 6.850mMGlycerol10%EDTA12.5 mM SDS2%Bromophenol blue0.02%β-mercaptoethanol1%Running buffer for western blottingGlycine14.4 g/lTris base3.05 g/lSDS1.0 g/lTransfer buffer for western blottingGlycine14.4 g/lTris base3.03 g/lThe above salts were dissolved in 800 ml of milliQ water and 200 ml of methanol was then added. The buffer was chilled before use.TBST buffer for Western blot10X of TBS (1000ml)Sodium chloride80 gPotassium chloride2 gDisodium hydrogen phosphate(Na2HPO4)14.1 gPotassium dihydrogen phosphate(KH2PO4)2.49 gMilliQ waterto 1000m

EDTA 2 mMThis was prepared at 50X concentration and used at 1X concentration. Both TBE and TAE were used as standard electrophoresis buffers.Gel loading buffer with dye Tris-Cl (pH 7.5) 250 mM Bromophenol blue/ Xylene cyanol 0.02% Glycerol 20%INOUE (PIPES) Buffer PIPES (Free acid) 10 mM CaCl2.2H2O 15 mM KCl 250 mM MnCl2.4H2O 55 mM pHwas adjusted to 6.7 with 1N KOH. PIPES gets into solution when the pH is greater than 6.7. MnCl2 was dissolved separately and added with stirring. The pH was then adjusted to 6.7 and solution wasfilter sterilized and stored at –20ºC.Z Buffer (for β-galactosidase assay) Na2HPO416.1 g NaH2PO45.5 g KCl 0.75 gMgSO4.7H2O 0.246 g H2O to 1000 ml pH was adjusted to 7.0 and stored at 4ºC.Pre-Hybridization Buffer 20X Saline-sodium citrate (SSC)3ml50% dextran sulphate2ml50X Denhardt’s solution1ml

20%SDS250 μl10 mg/ml Salmon sperm DNA100 μlDEPC waterto 10mlHybridization Buffer Same as pre-hybridisation buffer but contains the radio-labelled probe.SDS sampleBuffer(1X)Tris-HCl, pH 6.850mMGlycerol10%EDTA12.5 mM SDS2%Bromophenol blue0.02%β-mercaptoethanol1%Running buffer for western blottingGlycine14.4 g/lTris base3.05 g/lSDS1.0 g/lTransfer buffer for western blottingGlycine14.4 g/lTris base3.03 g/lThe above salts were dissolved in 800 ml of milliQ water and 200 ml of methanol was then added. The buffer was chilled before use.TBST buffer for Western blot10X of TBS (1000ml)Sodium chloride80 gPotassium chloride2 gDisodium hydrogen phosphate(Na2HPO4)14.1 gPotassium dihydrogen phosphate(KH2PO4)2.49 gMilliQ waterto 1000ml

1litreof 1X TBS +1 ml of Tween-2040% Acrylamide solution (29:1) Acrylamide39 g Bis-acrylamide 1 g H2O to 100 ml7.5M Urea 10%acrylamidecomposition 40% Acrylamide 12.5mlUrea22.5g5X TBE 10ml DEPC treated H2O to 50ml The gel mixtures were filtered through a 0.45 μ Millipore filter before adding APS and TEMED

Citrate Buffer Citric Acid (0.1 M) 4.7 volumesSodium citrate (0.1 M)15.4 volumesTE Buffer Tris-Cl (pH 8.0) 10 mM EDTA 1 mMTBE Buffer Tris-Borate 90 mM EDTA 2 mMThis was prepared as 5X solution and used at 0.5X concentration.TAE Buffer Tris-Acetate 40 mM

Buffers and solutions

ZFIN_ZDB-GENO-150320-5

ZFIN_ZDB-GENO-140811-5

RRID:ZFIN_ZDB-GENO-151014-5

ZFIN_ZDB-ALT-110408-5

RRID:ZFIN_ZDB-ALT-170606-5

the manuscripts that were discovered nine years ago, now in the University of Arkansas library with many of her other papers, are mostly complete and easily performed.

The children were exhibiting what I would call dramatizing, talking back, in serting, and taking over. Ballenger called this "entering in"

as one way of personalizing the stories, of drawing the sto ries to themselves; more important, it allowed them to control and manage plots and characters. T

Thus, talk ing back to the story and addressing characters directly begins to blur the distinction between the story world and the children's world

his spontaneous dramatization demonstrates participation in the story by imitating and physically interpreting what is going on in it

and his whole heart was filled

With the control of our public lands in our own hands, we can attract the tide of emigration, retain our own people in the country, and advance in prosperity as rapidly as the other provinces.

We connect Cologne´s established companies with global innovation and startups

Cologne is one of Europe’s leading medical centres. The health sector in Cologne stands out with a high level of expertise and top-rate cutting edge medicine. The medical fraternity in Cologne is made up of renowned medical experts at the cutting edge of their profession. Many of them have trained abroad and are members of national and international societies, chambers and research communities in their various disciplines. Similarly, the therapeutic, nursing and other skilled staff are trained and qualified on the highest scientific level. The profile structure in Cologne consists of 20 hospitals, clinics and highly specialized day and specialist clinics with more than 7,100 beds. The more than 2,200 doctors and 10,000 therapeutic, nursing and other skilled staff offer a wide range of experience, treating more than 300,000 patients every year from Germany and all over the world, on a residential and out-patient basis. The various hospitals and clinics work together in close cooperation. Specialists in various disciplines join together in advising the patient on the best possible treatment in each specific case; it goes without saying that this can also take place in the presence of an interpreter or doctor from the foreign patient’s home country.

The Cologne-based TÜV Rheinland headquarters is revitalizing its approximately 100,000 square meter business park with ten buildings in Poll. The management of TÜV Rheinland Immobiliengesellschaft mbH & Co. KG has developed an innovative concept with the engineering experts from Drees & Sommer as energy designer, building physicist and TGA planner: In future, there should only be one energy center. All buildings in the property are supplied with heat and cooling via the power grid of the new energy center. For heat supply, hybrid energy sources are used. These consist of the renewable raw material wood, a wood pellet boiler plant, as well as the fossil energy natural gas, gas condensing boilers and an integrated combined heat and power plant. The cold is generated by free-cooling, high-efficiency compression machines and absorption chillers. This can save 30 percent of primary energy compared to today. In addition, CO2 emissions will be reduced by more than 30 percent. The overall concept is modular in design and adaptable for the future.

evohaus innovative settlements in general evohaus irq (Intelligent Residence Quartiere) Settlements cover your heat demand primarily environmentally friendly and cost-effective by the sun. The need for heating is already low due to the good insulation of the evohaus architecture anyway. Remaining heat demand is covered by solar power. The solar power drives heat pumps that produce about three kilowatt hours of heat energy for heating or hot water with one kilowatt hour of electrical energy. The settlement gets its heat independent of gas, coal or other fossil fuels. The heat pumps are preferably switched on when enough solar power is generated. Water tanks store excess heat and provide the settlement with sunless times. An energy management system monitors and controls storage tanks and heat pumps. The evohaus irq concept is taking the step from a passive house to an active house: it not only saves energy but also generates electricity itself and uses it with intelligence.

"Green tires" reduce the fuel consumption of vehicles in urban traffic by up to seven percent (on average by 4.1 percent) and can save fleet operators thousands of euros in costs each year. In addition, these high-performance tires significantly reduce the CO2 emissions of vehicles compared to standard tires. These are the results of a joint tire test carried out by LANXESS, the world's leading manufacturer of synthetic high-performance rubbers for the tire industry, together with energy supplier RheinEnergie. RheinEnergie has therefore decided to gradually convert its vehicle fleet to "green tires". Initially, around 130 vehicles will be retrofitted as part of the usual wear change. For half a year, under real conditions, the fuel consumption of six identical RheinEnergie service vehicles in Cologne and the surrounding area was compared with both "green tires" and standard tires, thus determining the potential for savings. The vehicles with a weight of around two tons had comparable areas of application in the city of Cologne and the surrounding area during the test period. Driver, load weight and tank operations were identical for the vehicles. Over the entire test period, all six vehicles together covered a distance of around 37,000 kilometers. The result: The maximum fuel saving was 6.96 percent and a lower CO2 emission of up to 155 kilograms per 10,000 kilometers.

KVB cycle hire Smart mobility   Smart mobility is climate-friendly, sustainable, space-saving and networked. It relies on diversity and multimodality. The resident of a smart city does not remain loyal to one mode of transport. The result is a mobility patchwork that is tailored to the individual circumstances and that can be configured quickly and easily at any time. Energy-efficient and space-saving mobility has priority here. "Sharing" is smart! The sharing of things and information already establishes itself under the term "sharing economy" and places the function before the property, in order to use existing resources more efficiently. Smart mobility in urban areas is therefore primarily a matter of sharing a networked mobility offer from buses, trains, bicycles and cars. Smart mobility is not just a technological task. Especially in the inner cities, walking and cycling will provide space for quality of life and urban development through active mobility. This is where the bicycle rental system of the Cologne Transport Company (KVB) comes in by closing a gap in the combination of environmentally conscious and mobility-active mobility. The bicycle rental system of KVB stands for an open architecture. It is therefore not a system with only fixed station terminals after the well-known role models from other major cities, because a template for all cases, the complex events of a city can consider insufficient. The system offers users fully flexible rental and return in the street, but also stationary station terminals depending on the available options and needs. The rental terminals cover the entire span between conventional stations and purely virtual stations.  

The diesel exhaust gases of the Rhine ships pollute the Cologne air with pollutants and fine dust and the climate with a significant amount of CO 2 . A part of it does not arise during the journey, but while the ships are at anchor. Because their generators must also run to generate the necessary electricity. Here, "Landstrom" provides a remedy: Since 2015, RheinEnergie has gradually been equipping a large part of the moorings along the Rhine with uniform power connections. Consequence: During the lay times the ship diesels can be turned off.

CO2-free parcel delivery for Cologne Electric street scooters for the DHL

Cycling is active climate protection and pollutes cities much less than the rest of the road. With this in mind, the company has developed and offers cyclists from all over Germany the opportunity with the help of the Radbonus app to receive financial rewards from kilometers driven by countless partners such as health insurances, employers, online shops and many more. The company, which has been operating since October 2015, would like to reward and acknowledge the valuable contribution every single cyclist makes to the environment and to climate protection. " Cyclists are heroes of everyday life for me,"Radbonus founder and CEO Nora Grazzini comments. Born in Cologne, she describes herself as a passionate cyclist and believes in making the world a whole lot better with her business idea. After a distance of 50 kilometers, the first rewards can be erradelt.

Electric cars are an energy-efficient and potentially regenerative alternative to cars powered by fossil fuels. In order to promote this regenerative alternative, colognE-mobil has already installed 122 charging stations for electric cars (TankE) in and around Cologne, one of which is located on the Klimastraße in the car park behind the Kaufhof. Further charging points will soon be created directly on the Klimastraße.

With evopark, the entire parking process runs without cash or contact. The parking time is recorded digitally. Billing is convenient and collected at the end of the month. Another advantage: With the app you also keep the parking time always in view. If you would like to use the new offer, you can register online at http://www.evopark.de/ . The personal parking card comes within a few working days by mail.

As well as energy-saving lighting, Smart Home is an important building block for an energy-efficient and comfortable future. With smart homes and smart meters in the network, homeowners and store owners can reduce their electricity and heating costs by an average of 7%! Add to that the great comfort of making the apartment burglar-proof and controlling almost every aspect of heating, electricity or security in the building. So you can control from your smartphone whether the stove is still on at home, a window has been left open, the heating is running at full speed or the light is on. In addition, before the house is on fire, modern, networked smoke detectors report any alarm directly to the owner's smartphone. It can automatically be initiated various steps, such. B. that the fire department is called. In order to test some scenarios and saving opportunities in everyday life and to make known the possibilities offered by these modern technologies, Smart Home applications were installed on the Klimastraße in nine private apartments of the Nippes Tower and in the bookstore Neusser Straße. This was financed by the project Klimastraße or the company RocketHome . In addition, it is planned to equip the entire climate road with smart meters from RheinEnergie.

Along the Klimastraße, the street lighting was replaced by modern and elegant LED street lamps by the end of January 2014. From now on, about 55% energy and about 5 tons of CO2 are saved. The lighting in the shops and businesses on the Klimastraße is on all business days up to 12 hours on. Therefore, all owners were asked by the project to participate in a retrofit campaign on LED lighting. Even businesses and businesses from Cologne and the surrounding area of ​​Cologne have become aware of this campaign on the Klimastraße. In a temporary action, these are supported by small grants from the project SmartCity Cologne to equip their business premises with LED lighting.

In the framework of the project "Celsius" we investigate which method leads to the best possible results in order to increase the chances of realization. For this purpose, demonstration plants were built at three different locations in the city. In Cologne-Wahn and Cologne-Mülheim, the heat is extracted directly from the sewer using so-called gutter heat exchangers. The heat exchangers with a length of 60 and 120 meters are installed at the bottom of the canal. The heat transfer medium transports the heat from there to the heat pumps with a capacity of 150 or 200 kW in the boiler rooms of the schools supplied. In Cologne-Nippes, a total of three schools and a sports hall are supplied by sewage heat. Here, the wastewater is pumped through a newly laid, 400-meter-long bypass to the boiler room of the Edith Stein-.Realschule. There, in the largest direct evaporator in Germany (400 kW), heat is transferred directly to the heating circuit of the schools. With the three demonstration plants, an environmental relief of a total of 500 t CO2 / year is achieved. The use of wastewater heat is technically mature and well developed. Nevertheless, this form of waste heat utilization has so far been a niche existence. This is partly because it is still little known, often the necessary information is not available locally, their implementation is relatively complex and requires high investment. Further reducing these barriers is the goal of the Cologne CELSIUS project.