Binding Buffer (10X)

EMSA Buffer

Nuclear lysis buffer

Polydeoxy (Inosinate-cytidylate) (Poly dI-dC)

For Electrophoretic mobility shift assay (EMSA)

TBST

Dithiothreitol (DTT)ComponentsFinal concentrationFor 5 mlDTT1.0M0.7725gH2Oq.s

Materials and Methods462.2.5 Cell proliferation assayThe 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.2.2.6 Preparation of whole cell, cytoplasmic and nuclear lysatesIn order to extract the total cell homogenate, the culture media was removed and cells were washed with ice cold PBS. The cells were then gently scrapped and pelleted by centrifugation at 3000 rpm for 3 minutes at 4°C. The cell extraction buffer was added to the cell pellet and placed on rotor kept in cold room for 30 minutes for cell lysis. Lysed cellswere then centrifuged at 13000 rpm for 10min at 4°C. The supernatant was collected as cellular lysate. Protein concentration was estimated using Bradfordassay, described below.For a typical Western blot assay, 30-70μg of protein was loaded on theSDS-PAGE.For the preparation of cytoplasmic lysate,ice-cold hypotonic cytoplasmic extract buffer was added in the cell pellet andgently mixed with the pipette in a microfuge tube. The cell suspension was incubated on ice for 30 minto allow them to swell.After incubation, freshly prepared 10% NP-40was added andvortexed vigorously for 15 seconds torupture the plasma membrane. The contents were then centrifuged at 13000 rpmfor a minuteat 4°C and supernatant containing the cytoplasmic lysate was transferred to another pre-chilled microfuge tube and stored at –70°C.The pellet was then further processed for extraction of nuclear lysate.For this, ice-cold nuclear extractbuffer was added to the pellet and incubated on ice for 45 min with intermittent vortexingafter every 10 min of incubation. Finally, cell suspension wascentrifuged for 5 min at 14000rpm. The supernatant containing nuclear lysatewas stored at –70°C for further experiment

Preparation of whole cell, cytoplasmic and nuclear lysates

For preparation of Ultra competent cells

Inoue buffer

(f) Running buffer

Yeast weregrown in YPD (Difco) overnight,and sub-cultured at 0.2 OD600. Cells were harvested at 0.6-0.8 OD600. 1 OD600of each culture was used for the labelling. Cells were washed in SC-Metmedium twice, suspended in SC-Metmedium containing 25μCi/mLof 35S Met-Cys and pulsed for 15 min. Cells were washed twice in methionine-free medium and suspended in 300Lof Tris-saline. Cell suspension was counted in a liquid scintillation counter (Perkin Elmer-Tricarb 2900). The cpm values obtained were plotted using GraphPad Prism5

35S-Met uptake assay

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

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

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

20 mM HEPES500 mM NaCl 2 mM EDTA1% Triton-XYeast protease inhibitor cocktail and phosphatase inhibitor cocktail (added fresh to the buffer C)IP7 reaction buffer(10X)250 mM HEPES,pH 7.4500 mM NaCl60 mM MgCl210 mM DTT (1 M stock was made separately, aliquoted into 100 μL and stored at -20oC).10X buffer was made and stored at 4oC. An appropriate amount was added to the reaction mix to get a final concentration of 1X.DTT was added fresh to the reaction buffer just before use

Buffer C

2 mM EDTA5 mM DTT1% Triton-XYeast protease inhibitor cocktail and phosphatase inhibitor cocktail (added fresh to the buffer B)

20 mM HEPES pH 6.8100 mM NaCl

Buffer B

20 mM HEPES pH 6.8100 mM NaCl2 mM EDTA5 mM DTTYeast protease inhibitor cocktail and phosphatase inhibitor cocktail (added fresh to the buffer A)

Buffer A

Buffers for protein purification and IP7reaction

Buffer A1 mM EDTAin HPLC grade water (Fisher Scientific)Buffer B 1 mM EDTA(NH4)2HPO41.3 M, pH 3.8171.6 g of (NH4)2HPO4was dissolved in 750 mL of HPLC grade water. pH was adjusted to 3.8 with 75 mL of H3PO4by continuous stirring and the volume was made upto 1000 mL.Both buffers were filtered througha0.22 μm filter (Millipore) using vacuume filter apparatus (Tarsons) and degassing was performed atleast for 20 min using a vacuume pump

Buffers for IP7 purification

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

Volume was adjusted with water to 1 L and solution was sterilized by autoclaving.Pre-hybridization/hybridization buffer (Modified Church and Gilbert buffer)0.5 M phosphate buffer (134g of Na2HPO4.7H2O,4 mL of 85%H3PO4), pH7.27% (w/v) SDS10 mM EDTA Volume was adjusted to 1 L with DEPC treated sterile water. Buffer was aliquoted into 50 mL RNase free conical tubes (Corning) and stored in -20oC.Post hybridization wash buffersWash buffer 1 2X SSC 0.1% SDS Wash buffer 2 1X SSC 0.1% SDSWash buffer 3 0.5X SSC0.1% SDSBuffers were prepared with sterile DEPC treated water

TMN buffer10mM Tris-HCl, pH 7.45 mM MgCl2100 mM NaCl Permeabilization buffer950 μLof coldwater50 μLof 10% (wt/vol)sodium N-lauroyl sarcosineTranscription assay buffer(100 μL)50mM Tris-HCl, pH 7.4100mMKCl5mM MgCl21mM MnCl22 mM dithiothreitol 0.5mM ATP 0.25 mM GTP0.25mM CTP10mM phosphocreatine2.4 units creatine phosphokinase100μCi [α-32P] UTP (3,000Ci/mmol)Alkaline denaturing solution for DNA for membrane preparation0.5 M NaCl 0.25 M NaOH Volume was adjusted to 20 mLwith sterile water. Saline Sodium Citrate (SSC) buffer (20X) 3.0 M Sodium chloride 0.3 M Sodium citrate

Buffers fortranscription run on analysis

50 mM Tris-HCl,pH7.450 mM NH4Cl12 mM MgCl21 mM DTT0.1%DEPC37% sucrose solution

100mM NaCl30mM MgCl250μg/mLcycloheximide 200μg/mL heparin All the components were made in DEPC treated water.Gradient buffer10% sucrose gradient buffer50 mM Tris-HCl,pH7.450 mM NH4Cl12 mM MgCl21 mM DTT0.1%DEPC10% sucrose solutionTo analyse individual ribosome subunits, MgCl2 was eliminated from the gradient buffer.30% sucrose gradient buffer50 mM Tris-HCl,pH7.450 mM NH4Cl12 mM MgCl21 mM DTT0.1%DEPC30% sucrose To analyse individual ribosome subunits, MgCl2 was eliminated from the gradient buffer.50% sucrose gradient buffer50 mM Tris-HCl,pH7.450 mM NH4Cl12 mM MgCl21 mM DTT0.1%DEPC50% sucrose To analyse individual ribosome subunits, MgCl2 was eliminated from the gradient buffer.37% sucrose gradient buffer

Lysis buffer10mM Tris, pH7.4

Buffers for ribosome and polysome analysis

30%GlycerolMade in 100 mL.RNA sample loading buffer (10X)50% glycerol10mM EDTA 0.025% Bromophenol blue 0.025% Xylene cyanolInoue transformation buffer, pH 6.7(125 mL, prepared just before use)10 mM PIPES 15 mM CaCl2.2H2O 250 mM KCl 55 mM MnCl2.4H2O (1.361 g is dissolved in 10 mL of water separately)PIPES(0.307 g), CaCl2.2H2O (0.275 g) and KCl (2.325 g)were added to 80 mL ofsterile water while mixing with a magnetic stirrer and the pH was adjusted to 6.8with 1 N KOH. After attaining the appropriate pH, MnCl2solution wasadded slowly in aliquotes of 300 μL over 10 min,while stirring to avoidabrown precipitate.MOPS buffer(10X)0.2 M MOPS, pH 7.220 mM CH3COONa10 mM EDTABuffer was made in DEPC treated waterYeast transformation reagents1 M Lithium acetate 50% Polyethylene glycol2 mg/mLSalmon sperm carrier DNA Dimethyl sulfoxide (DMSO) Zymolyase cocktail buffer for yeast colony PCR 2.5 mg/mLZymolyase (ZymoResearch)1.2 M SorbitolZymolyase buffer was prepared in 1X PBS

Yeast lysis buffer for genomic DNA extraction50 mM Tris-HCl,pH 8.010 mM EDTA 150 mM NaCl 1% Triton-X 1% SDSAE buffer for RNA extraction50 mMSodium acetate,pH 5.31 mMEDTA,pH 8.0Solution was made in DEPC treated water. 0.2%diethyl pyrocarbonate (DEPC)was added to the water and stirred for 12 h. To remove DEPC,water was autoclaved twice. DNA sample loading buffer (6X)15.25 mg Bromophenol blue15.25 mg Xylene cyanol

Buffers for extraction and analysis of genomic DNA and RNA

EDTA (pH 8.0)186.1 g of EDTA.2H2O was dissolved into 800 mL of water stirredvigorously and the pH was adjusted with NaOH pellets. When the pH of the solution reached8.0 EDTA dissolvedcompletely and was made upto 1000 mL with water.Tris-HCl buffer (1M)121.1 g of Tris base was dissolved in 800 mLof water and pH was adjusted to 7.2 using concentrated HCl Tris-EDTA (TE) buffer 10 mM Tris-HCl, pH 8.01 mM EDTA Tris-Acetic acid EDTA (TAE) buffer 40 mM Tris base 1mMEDTApH was adjusted to 8.4with glacial acetic acid. TAE buffer was prepared as a 50X stock solution and used at 1Xconcentration.Tris-Saline20 mM Tris-HCl, pH 7.20.9% NaCl

PhosphateBuffered Saline (PBS) 137 mM NaCl 2.7 mM KCl 10 mM Na2HPO42 mM KH2PO4pH was adjusted to 7.3 using HCl and NaOH beforeautoclaving. PBS was prepared as a 10X stock solution and diluted to 1X concentration before autoclaving

Common buffers

Buffers and solutions

Plasmids containing the shRNA of interestwere either transfected transiently or were stably transfected. Transient transfection of shRNA was performed using eitherLipofectamine 2000 or PEI (as per the method explained before). Stable integration of shRNA was performed by transfecting shRNA along with retroviral packaging vector PCL-Ampho into BOSC23 packaging cells. The supernatantcontaining the packed viruses (viral medium)was collected at 48 and 72 hours of transfection. The viral mediumwas then added to thetarget cells in the presence of polybrene (8μg/mL). Two days later, cells were cultured in medium containing puromycin for the selection of stable clones.The clones stably expressing the desiredshRNA were identifiedandverified through western blotting and immunostaining using specificantibodies. A similar protocol was used to generate stable cell lines that expressed control shRNA

ShRNA

Cells were plated in a manner that they were 30-50% confluent on the day of transfection.Cells were washed with serum-free medium,and the serum-free medium was added to the cells as per plate size. SiRNA was diluted in the serum-free medium, and oligofectamine was diluted in serum-free media, separately (Table 10). Both the complexes were incubated at room temperature for 5 min. Diluted siRNA wasmixed gently with diluted oligofectamine and incubated at room temperature for 15 min. The final transfection mixture was added dropwise to the cells and mixed properly by gentle rocking. Cells were incubated for 4 hrs.,and the growth medium containing 10% FBS was added to the plates without removing the previous medium. Cells were incubated overnight at 37°C in a CO2 incubator. After overnight incubation, the siRNA transfection was repeated using the same protocol. Cells were harvested after 24-48 hours of second round siRNA transfection. The knockdown was detected bychecking the protein levels throughwestern blotting. (Note: SiRNA transfection is carried out in antibiotic free medium)Table 10: SiRNA transfection methodology

SiRNA

RNA interference

In planta growth assay for different strains of Xanthomonas oryzaepv. oryzicolawas performed by counting CFUs. For getting the CFUs, 1 cm2 leaf area surrounding the site of inoculation was cut and surface sterilized by dipping the leaf in 1% (vol/vol) sodium hypochlorite for 2 min followed by three washes with sterile water. To get the CFUs, sterilized leaves were crushed using mortar and pestle, and diluted appropriately for plating on PSA plate containing suitable antibiotics for differentially marked strains

In plantabacterial growth assay

strain or the ∆rpfFmutantharboring the Wild-typeallele in plasmid (pSC9).Genes that were significantly up regulated by 0.6 or more or down regulated by -0.6 or less fold (log2–fold change) were identified.The microarray data have been deposited in the NCBI Gene Expression Omnibus (GEO) under the GEO series accession number GSE53255

8x15k (AMADID: 25096) custom Agilent platform comprised of coding sequences for the three strains of Xanthomonas-X. oryzaepv. oryzae(KACC10331), X. oryzaepv. oryzicola(BLS256) and X. axonopodispv. citri 306 gathered from National Center for Biotechnology Information (NCBI). A total of 8113 probes were designed wherein 2120 probes corresponding to genes of interest replicated three times on Agilent platform. Feature extraction software GeneSpring GX version 10.5.1 of Agilent and GeneSpring GX percentile shift normalization was used for data analysis. Genes that were significantly up or down regulated by more than 1.5 fold and less than 0.5 fold were identified. Hierarchical clustering was performed for the differentially regulated genes and classified based on functional category. Data are the average of two hybridizations from biological replicates of each sample andraw data sets for this study are available at the Gene Expression Omnibus database (Accession number –GSE217809). Likewise, Microarray analysis for Xanthomonas oryzaepv. oryzicolawas performed by isolating RNA from the strains grown under low-ironcondition. The labeled cRNA samples were hybridized on to a Genotypic Technology Private Limited designed 8x15k (AMADID: 41087) Agilent platform. Data extraction from Images was done using Feature Extraction software v 10.7 of Agilent. Data normalization was done in GeneSpring GX using 75thpercentile shift and normalization to specific samples. Differentially regulated genes were clustered hierarchically to identify significant gene expression patterns.Genes were classified based on functional category. Hierarchical clustering of DSF regulated genes in X. oryzaepv. oryzicola grown under low-iron conditions is based on similar expression profiles in ∆rpfFmutant vs either the Wild-typeBXOR1 strain or ∆rpfF(pSC9). Clustering analysis was performed using GeneSpring GX Software using Average Linkage rule with pearson uncentered distance metric. log2–fold change differences between the ∆rpfFmutant with either the Wild-typeBXOR1

Xanthomonas oryzae pv. oryzae strains grown in PS medium to an OD600of 1, were collected, washed once with 150 mM sodium chloride (NaCl) solution to remove excess EPS. RNA isolation was performed using Trizol method described above. After isopropanol precipitation, RNA was frozen at -80°C. Quality of RNA was examined by determining the RNA integrity number (RIN) before microarray analysis. Microarray experiments were performed at Genotypic Technology Pvt. Ltd., Bangalore.Briefly, a

Microarray analysis

shaking at 200 rpm. 1% of overnight grown culture was inoculated in 100 ml fresh PS medium and grown to obtain log-phase culture. Log phase Xanthomonas culture was kept on ice for 10-15 min, aliquoted in 50 ml pre-chilled centrifuge tubes and centrifuged at 4000-5000 g at 4°C for 10 min. Supernatant was discarded and pellet from each tube was gently resuspended in 10-20 ml sterile chilled water. Next, cells were harvested by centrifugation at 4000 g at 4°C for 10 min and supernatant was discarded. Harvested cells were washed twice and finally resuspended in adequate amount of prechilled sterile water. 100 μl of cell suspension was aliquoted in sterile 1.5 ml microcentrifuge tubes and kept on ice. For transformation, Xanthomonaselectrocompetent cells and appropriate amount of plasmid DNA was mixed, and kept on ice in laminar hood. This mixture was added to 1 mm electroporation cuvettes (Biorad) and tapped gently to allow the cells to settle properly in order to avoid air bubbles. Competent cells were electroporated (1800 V, 25 μF, 200 Ω, 1mm cuvette) followed by immediate addition of fresh PS broth in the cuvette, mixed properly and taken in the microcentrifuge tubes. Microcentrifuge tubes containing transformed cells were incubated at 28°C for 2 hours with continuous shaking for recovery. After recovery, cells were plated on specific medium with appropriate antibiotics and incubated in 28°C plate incubator

For electrocompetent cell preparation, single colony of desired Xanthomonasstrain was inoculated in 5 ml PS medium and grown overnight at 28°C

Xanthomonastransformation

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

For estimation of tannase activity the reaction mixture (4 ml) contained 1.0 ml of 1.0% tannic acid (prepared in citrate-phosphate buffer, pH 5.0), 2.0 ml of citrate-phosphate buffer (pH 5.0) and 1.0 ml of appropriately diluted culture supernatant. The reaction mixture was incubated at 40°C for 30 min in a water bath. The reaction was stopped by adding 4.0 ml of 2.0% BSA solution. In the control reaction, BSA was added prior to incubation. Now the tubes were left for 20 min,at room temperature, for precipitating the residual tannins and subsequently centrifuged at 10,000 rpm for 20 min. The end product, gallic acid thus formed was estimated by diluting 20 μl of the supernatant to 10 ml with DDW. Now, the absorbance at 260 nm was read against a blank (DDW) in a UV spectrophotometer (1601, Shimadzu Corporation, Japan). One unit of tannase: One tannase unit is defined as the amount of enzyme that releases 1 μmol of gallic acid from the substrate (tannic acid) per ml per min under standard assay conditions

In this method, tannase activity was estimated through spectrophotometric method by determining the concentration of the end product i.e., gallic acid, by estimating the absorbance at 260 nm. Reagents: •Tannic acid (1.0%): The solution was prepared by dissolving 1.0 g of tannic acid in 100 ml of citrate-phosphate buffer of the desired pH.•Bovine serum albumin (BSA): BSA (2.0%) was prepared in citrate phosphate buffer (pH 5.0)

Estimation of tannase activity (Deschamp et. al., 1983)

Overnight-grown C. glabratacells were freshly inoculated either in YNBmedium or YNBmedium supplemented with BPS (50 μM) or FeCl3(500 μM) and allowed to grow for 4 h at 30°C, 200 rpm. After 4 h growth, cells were spun down at 4,000 rpm for 5 min in a refrigeratedcentrifuge set at 4°C and total protein was isolated. For estimation of histone deacetylase (HDAC) activity, 40 μg of protein samples were taken and HDAC Fluorometric Activity Assay Kit (#10011563; Cayman Chemical Company, Ann Arbor, MI, USA) was used as per manufacturer’s instructions. Fluorescence intensity values obtained inthepresence of the HDAC inhibitor, trichostatin A, were subtracted from those of the samples without inhibitorand plotted as relative arbitrary fluorescence units

Estimation of histone deacetylase (HDAC) activity

Bacterial plasmid DNA was isolatedusing the QIAprep® spin Miniprep kit (QIAGEN, 27106). 10 ml of LB medium supplemented with appropriate antibioticswas inoculated withasingle bacterial colony and incubated at 37°C for 12-16 h. Cultures were spun down at 8,000 rpm for 5 min, supernatant was discarded and the cell pellet was processed to isolate plasmid DNA as per instructions given in the kit. DNA was eluted either in nuclease-free water or in elution buffer provided in the kit and stored at -20°C until use

Plasmid isolation

Bacterial transformation

Bacterial plasmid DNA was isolatedusing the QIAprep® spin Miniprep kit (QIAGEN, 27106). 10 ml of LB medium supplemented with appropriate antibioticswas inoculated withasingle bacterial colony and incubated at 37°C for 12-16 h. Cultures were spun down at 8,000 rpm for 5 min, supernatant was discarded and the cell pellet was processed to isolate plasmid DNA as per instructions given in the kit. DNA was eluted either in nuclease-free water or in elution buffer provided in the kit and stored at -20°C until use

Plasmid isolation

E. coliDH5α ultra-competent cells were used for all bacterial transformations. Briefly, frozen DH5α ultra-competent cells were taken out from -80°C freezerandthawed on ice for 15 min. DNA to be transformed was added to the bacterial cell suspension and incubated on ice for 30 min. For transforming ligation mixtures and plasmids,5-10 μl and 100-500 ng of DNA was used, respectively. Followed by 30 min incubation on ice, heat shock was given for 60-90 sec at 42°C in a water bath and cells were immediately kept back onice for 2 min. To this,1 ml of sterile LB medium was added and tubes were incubated inashaker incubator set at 37°C, 200 rpm for 45 min.Next, cells were spun down and resuspended in 500 μl of LB medium. About 100-200 μl of resuspended cells were plated on LB-agar medium containing appropriate antibiotics and incubated for 12-16 h at 37°C. Transformants were purified on LB-agar plates containing appropriate antibiotics andpositive transformantscarrying desired DNAwere verified by PCR, restriction digestion and sequencing analyses

Bacterial transformation

Stripping buffer100 mM β-mercaptoethanol2 % SDS62.5 mM Tris-HCl (pH 6.7)Final volume was madeto 250 ml with water

Transfer buffer (10 X stock solution)0.25 M Tris-HCl (pH8.0)1.92 M Glycine1% SDSThe stock solution was prepared asa10 X concentrate and was diluted to 1 X concentration prior to use.1X Transfer buffer (1 litre)200 ml Methanol100 ml 10X Transfer buffer700 ml WaterTris-Buffered Saline (TBS)50 mM Tris150 mM NaClFinal pHof the bufferwas adjusted to 7.4 with HCl.Blocking buffer5% Fat-free milk0.1% Tween-20Final volume was made to 100 ml with 1 X TBS.Wash buffer (TBS-T)TBS (1 X final concentration)0.1% Tween-20Final volume was prepared with water

Buffers for western blot experiment

2% DTTThe stock solution of SDS loading buffer was made asa4 X concentrateand was added to the protein sample to the final concentration of 1 X.SDS-PAGE running buffer0.25 M Tris-HCl (pH 8.0)1.92 M Glycine1% SDSThe stock solution was prepared as a 10 X concentrate and was diluted to 1 X concentration prior to use.Resolving gel mix (12%, 10 ml)3.3 ml H2O4 ml 30% Acrylamide:N,N’-Methylenebisacrylamide (29:1) mix2.5 ml 1.5 M Tris-HCl (pH 8.8)100 μl 10% SDS100 μl 10% Ammonium persulfate (APS)4 μl N,N,N′,N′-Tetramethylethylenediamine (TEMED)Stacking gel mix (5%, 3 ml)2.1 ml H2O0.5 ml 30% Acrylamide:N,N’-Methylenebisacrylamide (29:1) mix380 μl 1 M Tris-HCl (pH 6.8)30 μl 10% SDS30 μl 10% APS3 μl TEMED

2% DTTThe stock solution of SDS loading buffer was made asa4 X concentrateand was added to the protein sample to the final concentration of 1 X.SDS-PAGE running buffer0.25 M Tris-HCl (pH 8.0)1.92 M Glycine1% SDSThe stock solution was prepared as a 10 X concentrate and was diluted to 1 X concentration prior to use.Resolving gel mix (12%, 10 ml)3.3 ml H2O4 ml 30% Acrylamide:N,N’-Methylenebisacrylamide (29:1) mix2.5 ml 1.5 M Tris-HCl (pH 8.8)100 μl 10% SDS100 μl 10% Ammonium persulfate (APS)4 μl N,N,N′,N′-Tetramethylethylenediamine (TEMED)Stacking gel mix (5%, 3 ml)2.1 ml H2O0.5 ml 30% Acrylamide:N,N’-Methylenebisacrylamide (29:1) mix380 μl 1 M Tris-HCl (pH 6.8)30 μl 10% SDS30 μl 10% APS3 μl TEMED

Total cell lysis buffer (Homogenization buffer)50 mM Tris-HCl (pH 7.5)2 mM EDTA10 mM Sodium fluoride*1 mM Sodium orthovanadate*1 X protease inhibitor cocktail (Sigma, P 8215)** Were added fresh before use.SDS-PAGE30% acrylamide solution29 g Acrylamide1 g N,N’-MethylenebisacrylamideDissolved in 100 ml H2O10% Sodium Dodecyl Sulfate (SDS)10 g SDS in 100 ml H2OSDS loading buffer130 mM Tris-HCl (pH 8.0)20% (v/v) Glycerol4.6% (w/v) SDS0.02% Bromophenol blue

Buffers for protein extraction and separation by SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis)

Phenol solution saturated with 0.1 M citrate buffer (pH 4.3 ± 0.2)was procured from Sigma (P4682)

Buffer C100 mM Tris-HCl (pH 7.5)10 mM EDTA10% SDSPhenol:Chloroform:Isoamyl alcohol (25:24:1) solution25 ml Tris-equilibrated phenol (pH 8.0)24 ml Chloroform1 ml Isoamyl alcoholDNA sample loading buffer0.25% Bromophenol blue0.25% Xylene cyanol15% FicollStock solution of the loading buffer was prepared in water as a 6 X concentrate and was added to the sample DNA to the final concentration of 1 X.RNA isolation bufferAE buffer3 M sodium acetate0.5 M EDTA (pH 8.0)Reagents used for RNA isolation were prepared in DEPC-treatedwater and stored at 4°C. For preparationof DEPC-treated water,0.1 ml DEPC was added to 100 ml waterand kept overnight onamagnetic stirrer. Followingincubation,the solution was autoclaved to remove any traces of DEPC.Acid phenol solution

Genomic DNA isolation buffersBuffer A50 mM Tris-HCl10 mM EDTA150 mM NaCl1% Triton-X1% SDSBuffer B50 mM Tris-HCl (pH 7.5)10 mM EDTA1.1 M Sorbitol50 mM β-mercaptoethanol (Added freshbefore use)

Buffers used for nucleicacid extraction

10 mg/ml carrier DNA 5 μlAbove-mentioned reagents were added to prepare thetransformation mixture, and the volumes indicated wereused per transformation.500-1,000 ng of desired transforming DNA was added to this transformation mixture and final volume was adjusted to 360 μl with sterile water.Carrier DNA (Sonicated salmon sperm DNA, Stratagene, 201190) washeat denatured at 95⁰C for 10 min and transferred on ice before additionto the transformation mixture.43 μl DMSO was added to each transformation mixture before heat shock.Zymolyase cocktail buffer for yeast colony PCR2.5 mg/ml zymolyase (MP Biomedicals, 0832092)1.2 M SorbitolThe cocktail was prepared in sterile water

Tris-acetic acid EDTA (TAE) buffer40 mM Tris Base0.5 M EDTAFinal pHof the bufferwas adjusted to 8.5 with glacial acetic acid.TAE buffer was prepared as a 50 Xconcentrate and diluted to 0.5X concentration prior to use as agarose gel electrophoresis running buffer and to cast agarose gels.HEPES [4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid] buffer1 M HEPESFinal pHof the bufferwas adjusted to 7.5 with NaOH.HEPES was used as a buffering agent forpreparationof different pHmedium. Buffer was filter-sterilizedby usinga0.22 μm membrane filterand stored at 4°C.INOUE transformation buffer10 mM PIPES15 mM CaCl2.2H2O250 mM KCl55 mM MnCl2.4H2OFor preparation ofINOUE transformationbuffer,above-mentioned solutes were dissolved in appropriate amount in 800 ml of water and then 20 ml of 0.5 M PIPES(piperazine-1,2-bis[2-ethanesulphonic acid])(pH 6.7) was added. Final volume was adjusted to 1 litre with water, buffer was filter sterilized by usinga0.22 μm membrane filter and stored at -20°C. Stock solution of PIPES was preparedseparatelyby dissolving 15.1 gm of PIPES in 80 ml of water, pH was adjusted to 6.7 using 5 M KOH and volume was adjusted to 100 ml.Yeast transformation reagents1 M lithium acetate 36 μl50 % polyethylene glycol 240 μl

Phosphate-Buffered Saline (PBS)137 mM NaCl2.7 mM KCl10 mM Na2HPO42 mM KH2PO4Final pH of the buffer was adjusted to 7.3with 11.6 N HCland volume was adjusted to 1 Lbefore autoclaving.PBS was prepared as a 10X stock solution and diluted to 1 X concentration before autoclaving.Tris-HCl buffer0.5 M Trizma BaseFinal pHof the bufferwas adjusted to 7.6 using 11.6 NHCl.Tris-EDTA (TE) buffer10 mM Tris-HCl (pH 8.0)1 mM EDTATE buffer was prepared as a 10 X concentrate and diluted to 1 X concentration before use

Common buffers

Buffers and solutions

ncubated with appropriate Alexa-488 conjugated secondary antibodies diluted in PBST with 2% BSA at room temperature for 1 h (for dilutions refer to Table-2.3). The cover slips were washed with PBST, mounted on glass slides using Vectashield mounting medium with DAPI. Images were acquired on a LSM 510 META confocal microscope (Zeiss, LSM acquisition software, 63x 1.4 N.A. objective) at a 0.7 scan zoom to collect maximum number of cells per field. The number of foci in each nucleus was manually counted by changing contrast identically across all the samples. A minimum of 10 random fields were imaged per sample. Data are represented as average number of foci per nuclei.

Localization of the DNA damage response proteins γH2AX, Rad51 and BLM and mitotic marker H3S10 (Histone H3 phosphorylated on Ser10) upon genotoxic stress and recovery was analyzed by immunofluorescence, following hypotonic lysis. Cells were seeded on cover slips in 12 well plates at 10-15% confluenceand incubated overnight. Cells were treated with 0.5 mM hydroxyurea and 0.25 μg/mL neocarzinostatin for 12 h. Coverslips were washed twice with PBS and cells were incubated in hypotonic lysis buffer containing 10mMTrisHCl (pH 7.4), 2.5mM MgCl2, 1mM PMSF and 0.5% NP-40, on ice for exactly 8 min on a shaker at 50-60 rpm. Cells were washed twice on ice, with ice cold PBS for2 min, with continuous shaking. Fixation was carried out with ice-cold 100% ethanol (which hadbeenkept overnight in -20°C) for 4 min, shaking on ice. 500 μLof ethanol was added to each cover slip during fixation. Cells were washedthrice with 1 mL PBS containing 0.2% Tween-20 (PBST), for 8 min each,at room temperature with shaking.Nonspecific interactions were blocked by incubating the cells for 30 minwith 2% BSA diluted in 1X PBS. Cover slips were again washed twice with 1 mLPBST, incubated with appropriate primary antibody (Table-2.3) diluted in PBS + 0.2% BSA for 2 h at room temperature. 200 μLof antibodywasplaced directlyonthecover slips. Post incubation, the cover slips were washed thrice with 1 mL PBSTfor 3 min each and the cells were

Immunofluorescence

3.4 mL H200.63 mL 30% acrylamide solution (acrylamide:bis-acrylamide; 29:1)0.83 mL 1 M Tris (pH 6.8)0.05 mL 10% SDS0.05 mL 10% ammonium persulfate (APS)0.005 mL N,N,N',N'-Tetramethylethylenediamine (TEMED)Resolving gel solution (12%)10 mL3.3 mL H204 mL 30% acrylamide solution (acrylamide:bis-acrylamide; 29:1)2.5 mL 1.5 M Tris (pH 8.8)0.1 mL 10% SDS0.1 mL 10% ammonium persulfate0.004 mL N,N,N',N'-Tetramethylethylenediamine (TEMED)SDS-Running buffer(Tris/Glycine/SDS)25 mM Tris-Cl192 mM glycine0.1% SDSTransfer Buffer25mM Tris-Cl190 mM glycine20% MethanolMTT dyeMTT dye was dissolved in PBS at 5 mg/mL concentration. Filtered through 0.45 μm syringe filters, and stored in dark at 4ºC

Hypotonic lysis buffer10mMTris Cl (pH 7.4)2.5mM MgCl2, 1mM PMSF 0.5% NP-40Hypotonic bufferPrewarmed 0.075 M KCl Fixatives used in this study100% ethanol kept overnight at -20ºCfor immunofluorescence by prelysis protocol70% ethanol kept overnight at -20ºCused for PI based cell cycle analysisMethanol: glacialactetic acid (3:1) for cytogenetic analysis4% Paraformaldehyde-4 g of paraformaldehyde dissolved in 100 mL waterPI staining solutionPBS containing, 0.1% Triton X-1000.2 mg RNase 20μg propidium iodideReagents required for Tris-Glycine SDS-PAGESDS-PAGE 30% Acrylamide solution29 g of acrylamide and 1 g bi-acrylamide (29:1 ratio) dissolved in 100 mL water10% SDS10 g SDS dissolved and 100 mL waterLaemmli buffer40% Glycerol 240 mM Tris/HCl pH 6.8 8% SDS 0.04% bromophenol blue 5% beta-mercaptoethanolStacking gel solution (5%) 5mL

Buffers

The method followed was similar to that describedpreviously with slight modifications (Jinet al., 1992; Schleifet al., 1973).Overnightbacterial cultures were grown in LBand subcultured 1:500 in the same medium in a volume of 20 mlat 30oC.Cultures were induced with 1mM IPTG at A600=0.4. 0.9ml samples were aliquotedat time intervals of 0 sec, 20 sec, 40 sec, 1 min, 1.5 min, 2 min, 2.5 min, 3 min, 3.5 min, 4 min, 4.5 min, 5 min, 5.5 min and 6 min into 0.1ml of 1mg/ml ice cold chloramphenicol and the samples were put on ice. After sampling, 0.5ml of each culture was taken for β-galactosidase assay.Square root of β-galactosidase activity (activity at time Tt−T0) was plotted against time. In the graph, the point of inflection of the curve on the X-axis determines the rate of elongation of RNAP whereas slope represents the promoter clearance, lacZmRNA stability and factors affecting translation of lacZ(Burovaet al., 1995)

RNA polymerase elongation rate measurement

Non-stringent washes were carried out in 2XSSC and 0.25-0.5% SDS in DEPC water.Stringent washing was done in 1XSSC and 0.5% SDS in DEPC water. Washing was carried out at 55-56oC for 20 minutes. After washing, the blot was covered in the saran-wrap and exposed to the phosphoimager film. After the desired time of exposure, the filmwas then scanned in phosphoimager and the picture saved.The densitometric analysis of the bands was carried out as described in the section 2.2.3.7.Normalization of the signal intensities in northern blotting experiments using probe against tRNA(U73)Arg5was done as follows. The intensity of the tRNA(U73)Arg5signalin the WT or the parent strain in the absence of IPTG was taken as 1 and the relative change in the other strain/growth condition calculated. The value thus obtained was corrected using the change in the corresponding 5S rRNA intensity relative to that in the WT/parent strain in the absence of IPTG

Washingof the membrane, exposure and scanning

Oligonucleotides and PCR products were end-labelled using phage T4-polynucleotidekinase (PNK, New England Biolabs or Fermentas or Sigma) with 32P-γ-ATP. The radiolabelling reaction mixture (20μl) contained 1X of buffer provided by the company, 10 units of T4-PNK and 40μCi of 32P-γ-ATP. The reaction mix was incubated for 1 hrat 37ºC and the reaction was heat-inactivated at 65oC for 20 minutes. The labelled oligonucleotides and DNA fragments were purifiedby the Qiagen nucleotide removal kit. Labelling efficiency was checkedeither by using Geiger-Muller (GM) counter orusing liquid scintillation counter.For scintillation counting, 1μl of radioactive sample wasadded to the 5ml scintillation cocktail, and radioactivity count was determined in the 32P channel of scintillation counter (Perkin Elmer, Liquid Scintillation analyzer, Tri-Carb 2910 TR, USA). Liquid scintillation cocktail consists of 5g PPO (2,5-diphenyloxazol) and 0.3g POPOP (1,4-bis (5 phenyl 1,2-oxazole) Benzene, adjusted to a volume of 1L in toluene

Radiolabelling of oligonucleotides

Recombineering was performed as described in(Yuet al., 2000)for engineering the linear DNA on the chromosome. The oligonucleotide primers were designed to amplify the DNA cassette to be engineered. Oligonucleotidesused for recombination contained30–50nt homology at the 5ʹ endtothesequences at the target siteand 20nt homology tothe DNA cassette at the 3ʹ end. The DNA cassettefor recombinationwas generated by PCR and would contain30-50 bp homologiesto the target site. A strain with the target DNA and carrying a defective λ-prophage with gam,betaand exo genes (thatfacilitate homologous recombination)under the control of a temperature-sensitive λ cI-repressorwas grown at 30oC. At an A600of 0.4, the culture was shifted to 42oC for 15 minutes to express gam,betaand exo genes. Cells becomecapable ofrecombining linear DNA introduced into the cell by electroporation. 50-100ng ofamplified DNA cassettewas used for electroporation whichwas performed using theBio-Rad Gene Pulser set at 1.8 kV, 25 μF with Pulse controller of 200 ohms

Recombineering

white colonies were recovered and purified to give growth. If the mutation caused synthetic lethality then white colonies (that lack the shelter plasmid) would not be observed since plasmid loss would result in growth arrest. Therefore, lethality was inferred when either white colonies were not recoveredor were recovered but failed to purify further

To determine whether a particular mutation conferred lethality in the ppGpp0or ΔdksAbackground, an assay was devised based on the use of an unstable, easy to cure shelter plasmidpRC7, similar to that described previously(Bernhardt & de Boer, 2004). In the wild-type strain carrying pRC7, this plasmid can be lost at a frequency of 20-30% in the absence of the selection. However, this will not be seen if the plasmid loss leads to cell death. Since the plasmid pRC7 confers a lac+phenotype, in the absence of the selection plasmid loss can be visualized on X-gal IPTG containing plates as white colonies in a Δlac strain whereas the colonies that retain the plasmid will appear blue.In order to carry outsynthetic lethal screen in the ppGpp0or ΔdksAstrains, the spoT or dksAgenes cloned in pRC7 under the control of lacpromoter were used. Theseshelter plasmids,namely,pRCspoT or pRCdksA, respectivelywere transformed into the ppGpp0or ΔdksAstrain. To test the synthetic growth phenotypes, the mutations of the genes to be tested were introduced by phageP1 transductions. The resultingstrains were grown overnight in LBcontaining the antibiotic selection for the shelter plasmid and IPTG for expression of spoTor dksA, subsequently washedin minimal A medium and dilutions(usually 10−5or 10−6) of these cultureswere spreadon X-gal and IPTG containing plates without antibiotic selection for the shelter plasmid. The phenotypes of the white colonies in comparison with the blue colonies were noted. Viability of the strains was inferred when

Blue-white screening for viability or lethality phenotype

The antibiotics were used at the below concentrations (μg/ml) unless otherwise stated

Antibiotics

“It’s become like a thing people say when they can’t make their rent,” says Jenna, 22, a New York video-game designer. “ ‘Well, I could always just get a sugar daddy,’ ‘I guess I could just start camming,’ ” or doing sexual performances in front of a Webcam for money on sites like Chaturbate. “And it’s kind of a joke, but it’s also not because you actually could. It’s not like you need a pimp anymore. You just need a computer.”

In their absence, some airports have had to close checkpoints, as Baltimore-Washington International did over the weekend

Washington state Attorney General Bob Ferguson said Thursday that Motel 6 shared the information of about 80,000 guests in the state from 2015 to 2017. That led to targeted investigations of guests with Latino-sounding names, according to Ferguson. He said many guests faced questioning from ICE, detainment or deportation as a result of the disclosures. It's the second settlement over the company's practice in recent months.

ZFIN_ZDB-ALT-050512-6

ZFIN_ZDB-GENO-120217-6

RRID:ZFIN_ZDB-GENO-100427-6

RRID:ZFIN_ZDB-ALT-070423-6

ZFIN: ZDB-ALT-160921-6

Adipose tissue is no longer considered to be an inert tissue that stores fat. This tissue is capable of expanding to accommodate increased lipids through hypertrophy of existing adipocytes and by initiating differentiation of pre-adipocytes. Adipose tissue metabolism exerts an impact on whole-body metabolism. As an endocrine organ, adipose tissue is responsible for the synthesis and secretion of several hormones. These are active in a range of processes, such as control of nutritional intake (leptin, angiotensin), control of sensitivity to insulin and inflammatory process mediators (tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), resistin, visfatin, adiponectin, among others) and pathways (plasminogen activator inhibitor 1 (PAI-1) and acylation stimulating protein (ASP) for example). This paper reviews some of the biochemical and metabolic aspects of adipose tissue and its relationship to inflammatory disease and insulin resistance.

Say: Teach ye the Cause of God, O people of Bahá, for God hath prescribed unto every one the duty of proclaiming His Message, and regardeth it as the most meritorious of all deeds.

Wherefore, after I have abridged the record of my father, then will I make an account of mine own life.

In 2016,Microsoft launched Tay, an experimental artificial intelligence chat bot. Learning from interactions with Twitter users, Tay was shut down after one day because of its obscene and inflammatory tweets. This article uses the case of Tay to re-examine theories of agency. How did users view the personality and actions of an artificial intelligence chat bot when interacting with Tay on Twitter? Using phenomenological research methods and pragmatic approaches to agency, we look at what people said about Tay to study how they imagine and interact with emerging technologies and to show the limitations of our current theories of agency for describing communication in these settings.

The Service Card for the Stuttgart Services project is the first electronic ticket for e-mobility in and around Stuttgart. In the initial phase of the project, subscribers have been able since 2015 to use the Service Card as an electronic ticket. Marketed under the polygo brand, the project was funded by the German Federal Ministry for Economic Affairs and Energy until June 2016.As one of 40 projects included in Baden-Württemberg’s “LivingLab BWe mobil” e-mobility showcase, the Stuttgart Services project seeks to make access to e-mobility services as seamless as possible for customers traveling in the Stuttgart region, and to supplement them with further citywide offers. The Service Card will not only open up the city’s e-mobility potential; it will also integrate everyday aspects of urban life by serving as a library card, swimming-pool membership card, and payment card.

Stadtinfo Köln (City Info Cologne) is a research project financed by the German Federal Ministry of Research that centres around the collection of various traffic data to be distributed to diverse platforms including the Internet, portable devices such as PDAs and mobile telephones, in-car navigation systems and variable message signs throughout the city. The project was implemented over a four-year period from 1998 to October 2002 by 15 partners in co-operation with the city of Cologne at a cost of €16.1 million.

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

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.

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.

Neusser Straße in the district of Nippes shows what a future SmartCity could look like, because a section of the street becomes Cologne's climatic road. There, the most important energy projects are implemented. All facets of climate protection are taken into account: from optimal building insulation and maximum heat efficiency to charging stations for electric vehicles and low-energy street lighting. Klimastraße offers innovative companies the opportunity to test their new products and services in everyday life. If possible, companies finance their projects themselves, promising projects are funded from the project budget of RheinEnergie AG. Companies also gain additional value by exchanging valuable information and innovative ideas with other companies, including at climate road events. For all the enthusiasm for innovation, of course, only technology is used that meets the very strict German safety requirements. In addition, RheinEnergie and the City of Cologne make sure that the high Cologne supply standards are adhered to. For all new projects, safety comes first - technically as well as logistically. That is why not everything changes in the climate route - but certainly much better. The following section deals in more detail with the individual projects.

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 HOOU is a cross-university project, which is supported by the network of the six state-owned Hamburg universities * with the UKE, the Department of Science, Research and Equality, the Senate Chancellery and the Multimedia Kontor Hamburg (MMKH).

Fast and easy to shop or to go to the city center? This works very easy in Hamburg. With the Park and Joy app, drivers can quickly find, book and pay for free parking spaces - all via smartphone. Parking has never been so much fun!

Knowledge transfer at all levels Tutech combines science, business and society We know what is important in technology and knowledge transfer at the interface between university and industry. We speak both languages ​​- those of science and those of companies - and have been successfully combining entrepreneurial and scientific potential for 25 years. Our mission and goal is to create sustainable value through the application of new research results and inventions, and we do so by acting as a consultant, broker, initiator and coordinator at national and international levels. Tutech is a privately organized subsidiary of the Technical University of Hamburg and the Free and Hanseatic City of Hamburg. Together with our sister company Hamburg Innovation , we connect all public law schools of the city as well as numerous research institutions of the Hamburg Metropolitan Region.

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.

With the start-up garage, comdirect has consciously decided to focus on founders and their ideas at a very early stage. Thus, for the participation in the start-up garage initially only a basic idea necessary, the development of a prototype then takes place during the project phase in the context of the start-up garage. comdirect invites start-ups to pitch their space with FinTech ideas in the comdirect start-up garage. The ideas are evaluated for their impact and opportunities for the banking and finance industry. We offer intensive support to the chosen start-ups!

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

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

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

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.

In the eCulture Cloud, the digital cultural content of Hamburg will be stored in bundled form in the future. Among other things, this cloud offers the possibility of making (private) collections, libraries, image and video archives accessible to the public, even if they can not find a place in exhibitions of the institutions. The particular attractiveness of this project lies in the diversity of the collection contents. Because these are not only composed of historical documents, but also give deep insights into modern phenomena, such. B. in pop culture. In addition, it is not uncommon for creators themselves to start collecting collections or archives according to their personal needs

CityScopes are interactive, digital city models that analyze urban relationships and simulate development scenarios.They typically consist of model tables and "data blocks" on which information is projected. In this way, complex city data can be illustrated simply and transparently for concrete tasks and experimentally carried out as "what if" scenarios. CityScopes are particularly suitable for group discussions and participation workshops in which both professionals as well as laymen can participate. Multifunctional relationships can be displayed and changed quickly, with CityScopes providing fast visual feedback on potential impacts.

Hamburg Open Online University Los geht's!

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.

The integration of Internet of Things (IoT) into skill sets of intermodal traffic management is a big challenge. The results of this study will show the demand of qualification integrating smart technologies in this area of work and it will be possible to transfer these results to forecast future demand in smart cities in Germany, Europe and worldwide, with the help of our partners:

Smart lighting is situated at a pilot section in the port of Hamburg (Hohe Schaar Street): 4 km long; hosting 102 LED luminaries and 60 sensors. Smart Lighting provides follow-me-light and better safety for pedestrians and cyclists with the same lighting performance. Smart lighting combines detecting technologies of thermal sensors for the intelligent control. System includes Smart Traffic and Incident management: traffic monitoring and automatic incident detection.

The University Medical Center Hamburg-Eppendorf is the teaching hospital of the University of Hamburg and Europe’s most advanced paperless hospital. With about 10,000 employees, the UKE is the third-largest employer in the Free and Hanseatic City of Hamburg. About 2,400 of them are medical specialists and researchers, while more than 3,100 work as nurses and therapists. Together with its University Heart Center Hamburg and the Martini Clinic, the UKE has more than 1,730 beds.

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.

e-Mo­bi­li­ty in the port Elec­tric ve­hi­cles are be­com­ing in­creas­ingly com­mon­place in road trans­port. We are also re­view­ing ways of ex­tend­ing e-Mo­bi­li­ty to pas­sen­ger and freight traf­fic in the har­bour area. We are there­fore press­ing ahead with charg­ing in­fra­struc­ture, in col­lab­o­ra­tion with the op­er­a­tors of pub­lic charg­ing pil­lars. At the cruise ship ­ter­mi­nal, we plan to use pref­er­en­tial e-Ta­xis. In ad­di­tion, we are analysing the vi­a­bil­ity of e-Mo­bi­li­ty for our staff.

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.

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

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.

Funding programs for investment and innovation. Berlin offers attractive funding programs for all phases of a company’s development – from seed to growth financing. The range of financial incentives here extends from public loans to guarantees and venture capital, and even to direct grants for investment and innovation projects. What does the Business Financing Package offer? Do you intend to locate to Berlin, to grow at your current location, or are you planning an investment project? Our experts will find the right financing solution and will accompany you in your applications for funding. Here, we work closely with project management agencies, funding banks and potential financing partners on a state, national and EU level. How it works: We discuss the plans for your project, which ideally will already have been worded in the form of a project description or business plan. Together, we conduct a review of the conceivable funding and financing instruments, make contact with relevant partners and accompany you through the application process.

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.

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.

Berlin can attract by far the most risk capital nationwide and is becoming the most important target area for foreign investors. With a plus of almost 200%, the investment volume of venture capital in Berlin rose in just one year (2017) to almost 3 billion euros, putting Berlin just behind London, in second place. Local business angels, company owned and university related incubators, accelerators as well as national and international venture capital investors support Berlin’s young entrepreneurs in the foundation phase. Numerous successful start-ups have emerged that way, e.g. Zalando, SoundCloud, Wooga and Delivery Hero.

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.

Pro Fit – Programme to Promote Research, Innovation and Technologies In particular, the Pro Fit programme aims to enhance the technological competitiveness of small and medium-sized enterprises (SMEs) by intensifying research and development in individual and joint projects, and supporting the market launch of innovations. Pro Fit Innovation Funding: Company research projects and research institute joint projects in the areas of industrial research and experimental development Loans: Individual and joint projects in experimental development and production organisation, market preparation / launch by companies Pro Fit Early Phase Funding and Loans: Improving the potential sources of financing for newly established companies for developing and operating their business infrastructure, as well as forward and / or parallel financing to implement an innovation project.

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BSR’s MHKW Ruhleben Waste-to-energy power station with an annual capacity of 520,000 tonnes of waste forms the centrepiece of Berlin’s waste disposal. It went into operation in 1967, and has since been successively extended and refitted many times. With the com­missioning of the new Line A in September 2012 (together with the decommissioning of Lines 5 to 8), the MHKW now has a total of five incineration lines (Line A and Lines 1 to 4). It operates in a continuous three-shift system.

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

The 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.

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.

More cycling by the people of Berlin contributes to a better quality of life and environment in the city. Therefore the City of Berlin works hard to promote cycling as an alternative mode of transportation and to improve the conditions for cycling around the city.