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.  

The Cologne-based company Coptr Warn- und Schutzsysteme GmbH has been developing and producing innovative, precise, acoustically-optically smart on-site warning systems for several years.   Whether thunderstorms, hurricanes, extreme heat or pollutants in the air, the warning systems automatically warn and alert people in the open air to get to safety from lightning strikes and other high-threshold, potentially life-threatening weather and environmental hazards.

HamburgService - Kita-Infosystem

In January 2013, the Hamburg Social Impact Lab opened its doors. Since then, it has supported Hanseatic social entrepreneurs in a space of about 160 square metres. Social Impact Start programme scholarship holders receive coaching and consulting here, as well as plenty of further support to set up their social businesses. The Hamburg Social Impact Lab holds many events for interested persons on all aspects of social entrepreneurship

As a subsidiary of the  Hamburg Investment and Development Bank  , we support innovative business start-ups and young, innovative companies in Hamburg in order to strengthen the startup scene in Hamburg and to contribute to the development of promising companies. For this purpose, we have two ideal with InnoRampUp and the Innovation Starter Fund Hamburg

We turn ideas into enterprises Hanse Ventures is the company builder in Hamburg. We develop our own internet and mobile business concepts, and implement these together with suitable founder teams.

nextMedia.Hamburg is Hamburg's joint initiative for the media and digital industry.

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.

What is the transparency portal?The Transparency Portal Hamburg is the information register required by the Hamburg Transparency Act (HmbTG) , by means of which all information required to be published by law can be anonymously researched. It is the central access to up-to-date data and information of the Hamburg administration and provides a search over the full text of all data records in order to ensure easy findability of the searched content.

The CityScienceLab of HafenCity University Hamburg is exploring the transformation of cities in the context of digitization with partners from civil society, politics, business and science. It pursues a decidedly interdisciplinary and transdisciplinary perspective by linking technical issues with social and cultural developments (more under Team ). To make cities healthier, more liveable, and more efficient in the future, CityScienceLab uses urban data to develop new tools and digital city models (CityScopes). These new tools allow the visualization and simulation of complex urban developments and support urban actors in the decision-making process (more under Research and Teaching ).The city of Hamburg is the "Living Lab", in which urban change processes are comprehensively researched and developed right down to concrete applications. In addition, the CityScienceLab works in close cooperation with the City Science Group of the MIT Media Lab (Cambridge / USA).

The learning portal of the Center for Education and Training (ZAF) is aimed at the employees of the Free and Hanseatic City of Hamburg. The learning portal of the Center for Education and Training (ZAF) informs you about the continuing education offers of the ZAF and enables you to register online the events. Both the use of the platform, as well as the registration is free of charge for you

StadtRAD Hamburg - get on and off! The StadtRAD makes you spontaneous and individually mobile. Whether as a professional, leisure or tourist you experience Hamburg in a special way - very close to the pulse of the city. On many loan stations throughout the city, you have the option to rent a city bike around the clock and return it - as easy as cycling.

Start into the next Generation' is a project run by the Hamburg authority for schools and training. It was launched two years ago with the aim of improving learning outcomes by using technology in the classroom. Six pilot schools were selected to take part in the project with over 90 classes and 2,000 students involved. Each school was provided with a secure wireless network but was free to choose its pedagogical approach to using the technology. Students were asked to bring a laptop, tablet or phone to school (in 90% of cases they chose a smartphone). The schools were supported with training and with advice on legal and data protection issues (all parents were given a document to sign explaining what the devices would be used for). The Hamburg schools authority also provided a learning platform which gives access to digital materials and allows students to communicate, submit homework and complete self-assessments.

Apartimentum in Hamburg will be the smartest home in Europe with  44 apartments, all rented for a flat-rate, including all services – powered by Cisco IP technology.

Port Mo­ni­tor The con­trol room soft­ware, Port Mo­ni­tor, al­lows us to keep all the stake­hold­ers in the port of Ham­bur­g up-to-date. A va­ri­ety of in­for­ma­tio­n is cen­trally gath­ered and can also be ac­cessed re­motely, such as elec­tro­nic cards, ves­sel ­po­si­tio­ns, wa­ter level­ da­ta, berths, cur­rent con­struc­tion sites, planned dives and bridge heights and widths. Im­por­tant in­for­ma­tio­n is there­fore al­ways ac­ces­si­ble to all those in­volved on land and on the wa­ter.

Vir­tu­al de­pot Truck jour­neys with empty con­tai­ners put an un­nec­es­sary strain on the en­vi­ron­ment. We have there­fore de­vel­oped the so-called vir­tua­l de­pot to op­ti­mise the move­ment of empty con­tai­ners be­tween pack­ing ­companies. The cloud-­ba­sed sys­tem in­forms par­tic­i­pat­ing op­er­a­tors which con­tai­ners are to be de­liv­ered back to the de­pot. The pack­ing com­pany then re­quests these di­rectly. The re­sult: no more un­nec­es­sary empty trips to the de­pot.

Smar­t main­te­nance The in­fra­struc­ture in the port of Ham­bur­g is mon­i­tored us­ing mobi­le end de­vices, such as ta­blets or smart­pho­nes. When con­trolling roads, bridges and tracks, these de­vices au­to­ma­tically send mea­sure­ments to the down­stream IT sys­te­ms, where the da­ta is processed, stored and edited. The aim is to make the main­te­nance ­proces­ses more ef­fec­ti­ve and ef­fici­en­t and to im­prove the qua­li­ty of no­ti­fi­ca­tions.

In­tel­li­gen­t rail­way point Fre­quently used points on the har­bour rail­way are fit­ted with sen­so­rs that trans­mit da­ta to a cen­tra­l IT sys­tem in real-time. They col­lect a va­ri­ety of data by mov­ing or pass­ing over the switch­ing points and thereby pro­vide in­for­ma­tion about the con­di­tion and wear of the es­sen­tial op­er­a­tional in­ter­sec­tions. The ben­e­fit: we can iden­tify main­te­nance work or re­pai­rs at an early stage, thereby avoid­ing down­time.

Shore power from re­new­able en­er­gies Thanks to a land­side cruise liner power sup­ply sourced from re­newa­ble en­er­gi­es, we are sig­nif­i­cantly re­ducing the en­vi­ron­men­tal im­pact in Ham­burg. These ocean-go­ing gi­ants are sup­plied with elec­tric­ity via a trans­former ­sta­ti­on and mo­bi­le trans­fer mech­a­nism at the Al­to­na cruise ship ­ter­mi­nal. The di­men­sio­ns of the land­side power plant are unique in Eu­ro­pe. We are cur­rently con­sid­er­ing us­ing sim­i­lar mo­dels in other ar­eas of the port in fu­ture.

smart­PORT en­er­gy The HPA pro­motes en­vi­ron­men­tally-friend­ly mo­bi­li­ty and ad­vo­cates re­du­ced en­er­gy con­sump­tion. smart­PORT en­er­gy there­fore helps limit its de­pen­dence on con­ven­tio­nally gen­er­ated power, re­duce emis­sio­ns and save money. It fo­cuses on three core ar­eas: re­newable en­er­gi­es, en­er­gy ­ef­fici­ency an

Open Data Portal Munich The Open Data Portal is a platform for the provision of open administrative data of the City of Munich. Further information

Smart Cities: Stockholm Royal Seaport Project Update

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.

Berlin is Germany’s hotspot for founders and also the new venture capital. Entrepreneurs – and also potential entrepreneurs – find exactly the right environment here to implement their business ideas. With around 40,000 business registrations per year and more than 500 startup companies, Berlin is undisputedly Germany's founder capital and is expanding its nationwide lead. The capital is particularly appealing for founders in the creative sectors and technology. The starting conditions are advantageous: office and location expenses are much lower than in other major cities. Berlin attracts young, highly qualified people from all over the world. The high life quality at comparably low living costs, the vital scene life and international environment are the reasons for young entrepreneurs to implement their business ideas here. Numerous national and international studies regard Berlin as a leading global location for business start-ups with the world's best growth potential. The start-up scene is not only gaining importance as a job engine for the city, but has also become an important driving force for the Berlin office market.

Internationalisation programme – support for SME projects Support for small and medium-sized enterprises Do you have a small or medium-sized enterprise (SME) based or with a facility in Berlin and are you looking to enter new markets abroad for your products or services? In order to boost your international competitive strength, we can support you with a grant to open up new markets.

Co-working spaces are generally fully-equipped offices and working areas, which offer an opportunity to network within the “community”, alongside complete infrastructure with a range of services. They are used by individuals, startups and an increasing number of outsourced departments of established companies as their main place of work. For the user, they represent a flexible and, depending on the deal, cost-effective alternative to conventional office spaces in self-contained rental units. Startups and larger companies are increasingly preferring co-working spaces over classic office spaces, as they offer straightforward opportunities for an exchange with other people, or to recruit new employees. Berlin is the capital city of company founders. The range of well-equipped co-working spaces in a central location is very wide, and points to continued dynamic growth. The spectrum ranges from small units with a few tables to professionally operated offices with hundreds or sometimes even over 1,000 workplaces. International providers allow their members the opportunity to make use of desks and meeting spaces in other cities around the world.

With our online tool it is easy to find available financing options for your business in Berlin. Gain an overview over the relevant publicly funded programs and other funding opportunities and learn how to apply.  With just a few clicks you can find individual ways to finance your business in Berlin. The aim of this Funding Finder is to make it easier for founders and entrepreneurs in Berlin to access the various types of financing and funding.The range of funding opportunities includes not only public sources of funding but also other financing options such as venture capital, crowd funding, leasing and factoring.The information provided represents a selection of what in our experience are the most common forms of financing and funding which are available to businesses in Berlin. It is directed at current and future member companies of the CCI Berlin. 

Our web app* is the personal assistant for anyone wanting to live and work in Berlin. After answering just a few simple questions, you will receive a customised to-do list to help make your arrival in the German capital easier.Whether you are looking for work, planning to set up or relocate a company, or wanting to embark on training or study courses, the web app will be able to help you by providing specific tips and information along with the details of various contact partners, thereby making the first steps towards this new part of your life that bit easier.

Transfer BONUS Do you have a small or medium-sized company and are looking to develop your products and services further? Would you like to step up innovation and secure your company's future? And do you need the support of a scientific institution? Then you should make use of the Transfer BONUS. This support programme helps to finance co-operation between companies and scientific institutions from Berlin and Brandenburg.

Berlin Innovativ - Berlin innovation Our added benefit for your digital future and internationalisation Do you need financing for your project and do you meet with one of our innovation criteria? Under the 'Berlin innovation' programme, loans of up to EUR 2m are granted via your bank in order to finance investment and working capital. The advantage for you is that IBB provides your bank with 70% liability redemption. This support programme especially targets innovation companies and start-ups in Berlin with the aim of helping them to strengthen their competitiveness and to develop new markets.

Berlin is a high-tech location with a clear focus on cutting-edge technologies and knowledge-based services. BERLIN INNOVATION is a technology platform which showcases innovations “made in Berlin”. It serves as a "showroom" to establish Berlin as a major innovation hub and offers information about innovations to the wider public, businesses, investors and state-owned companies in Berlin. Clearly structured information on innovative products, processes and services from businesses in Berlin offers insights into innovations which are successfully implemented in the city. The aim of this information is also to stimulate demand for innovative solutions to business needs.

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.

Some 60,000 tonnes of organic waste are collected by BSR every year in the city’s BIOGUT bins. Since the summer of 2013, this organic waste has been treated in the newly-erected biogas fermentation plant in Berlin-Ruhleben. Recycling and energy recovery mean that in comparison with the composting procedures previously used it has been possible to reduce emissions of greenhouse gases. The biogas plant of BSR in Ruhleben has a capacity of 60,000 tonnes. The plant oper­ates using the dry fermentation method. Microorganisms from the organic waste generate the biogas. This method is particularly suitable for organic waste with a water content of 60–80%, which is typical for kitchen waste from Berlin households. After the biogas has been cleaned, treated and concentrated, it consists of 98% methane, and is therefore chemically identical with natural gas. After having been prepared in this way it can therefore be fed into the gas supply so that the natural gas vehicles used by BSR for waste collection can be refuelled at its own filling points. In the coming years it will be possible for BSR to operate as many as 150 natural gas vehicle

Pedestrians should have the possibility of getting through the capital without obstacles. With many measures our policy contributes to strengthen the pedestrians compared to the motorised traffic participants.