Activation of LSAX (strong anion exchange ) array

Overnight cell culture raised in LB medium was subcultured 1:100 in LB with 20 mM MgCl2. When the A600 reached 0.4-0.6, the culture was centrifuged at 2800g for 5 min at 4 ̊C. To the cell pellet 0.4 volumes of ice-cold TBF-I buffer was added and incubated on ice for 15 min. The cell suspension was centrifuged at 2800g for 5 min at 4 ̊C and the cells recovered were dissolved in 0.04 volume of ice-cold TBF-II buffer and kept on ice for 45 min. 100 μl aliquots of these competent cells were used for transformation using the normal transformation protocol

TBF method for preparation of high competency cells

For routine plasmid transformations, where high efficiency is not required, the following method which is a modification of that described by Sambrook and Russell (2001) was used. An overnight culture of the recipient strain was subcultured in fresh LB and grown till mid-exponential phase. The culture was chilled on ice for 15 min, and the steps hereafter were done on ice or at 4°C. The culture was centrifuged, and the pellet was resuspended in one third volume of cold 0.1 M CaCl2. After 15 min incubation on ice, the cells were again recovered by centrifugation, and resuspended in one tenth volume of cold 0.1 M CaCl2. The suspension (0.1 ml) was incubated on ice for 1 h after which DNA was added (~10-100 ng of DNA in less than 10 μl volume). The mixture was again incubated on ice for 30 min, and then heat shocked for 90 seconds at 42°C. Immediately 0.9 ml of LB broth was added to the tube and incubated at 37°C for 45 min for phenotypic expression of the antibiotic marker before being plated on selective medium at various dilutions. A negative control tube (with no plasmid DNA addition) was also routinely included in each of the experiments

Calcium chloride method

Transformation protocols

Antibiotics were used at the following final concentrations (μg/ml): Rich media Minimal media Ampicillin (for plasmids) 100 50 Ampicillin (chromosome) 30 30 Chloramphenicol (for plasmids) 50 25 Chloramphenicol (chromosome) 25 25 Kanamycin 50 25 Nalidixic acid 50 - Rifampicin 100 - Streptomycin 50 100 Streptomycin 100 200 Spectinomycin 50 100 Tetracycline 15 8 Trimethoprim (for plasmids) 60 30 Chloramphenicol 0.1mg/ml The 10 mg/ml chloramphenicol stock in ethanol was used to make 0.1mg/ml solution in water

Antibiotics

were then centrifuged at 1065 x g for 5 min at 4°C. The pellet obtained was resuspended in 1mL ice cold PBS and washed 5 times with PBS. Finally the pellet was resuspended in 1mL lysis buffer (10mM Tris-HCl containing 0.1% Triton X-100) and fluorescence was measured at 380nm/525nm. To normalize different samples and account for any errors in cell number between samples, 10011L of the above sample was also used for protein estimation, carried out as described above

Monodansylcadaverine (MDC) is a selective marker for autophagic vacuoles (Biederbick et al., 1995). It is an auto-fluorescent drug accumulates in acidic compartments by ion trapping and also is thought to interact with the membrane lipids of the vacuoles. Stock MDC (50mm, prepared in acetic acid) was diluted to a concentration of 50!lM in M199 medium containing 10%FBS. 107 parasites after appropriate treatment were resuspended in 1mL of working stock and incubated in the dark for 10 min at RT. These

Assay for measuring the level of autophagy in Leishmania parasites (MDC staining)

DNA fragments were resolved on 1-2 % agarose gel containing 0.5~-tg/mL ethidium bromide in Tris-Acetate-EDTA (TAE) buffer (40mM Iris-acetate, 2mM EDTA, pH 8.1). The samples were mixed with equal volume of 2X loading dye containing bromophenol blue, and the samples resolved by applying a voltage of 5-7 V /em. The resolved DNA fragments were visualized under ultraviolet illumination (312nm) and the relative band size was determined by comparison against DNA ladder with bands of known sizes. When required the images were acquired using a UVP Gel Documentation system

Agarose gel electrophoresis

5% glycerol)containing (i) 5′-end-labeled DNAfragmentof 1200 cpm radioactive count(ii) 1 μg each of bovine serum albumin andpoly(dIdC)(iii) the protein at the indicated monomer concentrations and (iv) when required,co-effectorsat specified concentrations. The reaction mixture was incubated at room temperature for 30-minsand the complexes were resolved by electrophoresis on a non-denaturing 5%polyacrylamide gel (39:1 acrylamide:bisacrylamide)in 0.5X TBE buffer pH8.3, at 12.5V/cm for 3 hrs at 18°C.The gels were then dried on a gel drier at 80°C for 45 minsand the radioactive bands were visualised with a Fujifilm FLA-9000 scanner.For DNA bending EMSA, co-effectors were not added in the binding reaction but at aconcentration of 0.1 mM in both the gel and running buffer

The DNA templates were obtained by PCR from E. coligenomic DNA. After 5-end labeling, the PCR fragments were purified by electroelution following electrophoresis on 6% native polyacrylamide gels (Sambrook and Russell,2001). EMSA reactions were performed in 20 μl reaction volume inEMSA binding buffer(10 mM Tris-Cl at pH 7.5, 1 mM EDTA, 50 mM NaCl, 5 mM dithiothreitol, and

Electrophoretic mobility shift assay (EMSA)

Typically 200-300 ng of DNA was used in each ligation reaction. The ratio of vector toinsert was maintained between 1:3 to 1:5 for cohesive end ligation and 1:1 for blunt endligation. The reaction was generally performed in 10 μl volume containing ligationbuffer (provided by the manufacturer) and 0.05 Weiss unit of T4-DNA ligase, at 16ºCfor 14-to 16-hrs. On using the rapid ligation kitfrom Fermentas, incubation was at 22ºC for 1-2 hrs

Ligation of DNA

purification of DNA fragments werefrom Qiagen or HiMedia. The oligonucleotide primers used in this study were mainly synthesised by Ocimum Biosolutions or MWG Biotech. The radioactive chemicals were procured from BRIT Mumbai

Chemicals were obtained from commercial sources. Most of the chemicals such as amino acids, antibiotics, sugars, IPTG, ONPG and X-gal were obtained from Sigma Chemical Co. The media components for the growth of bacteria were mostly from HiMedia laboratories. The materials used in the recombinant DNA experiments such as restriction endonucleases, T4-DNA ligase, DNA-polymerases and DNA size markers were obtained from companies including New England Biolabs, MBI Fermentas and Stratagene.RNA isolation chemicals like Reverse transcriptase, trizol, RNA loading buffers and dyes and RNA size markers were obtained from Invitrogen and Sigma. Protein markers were obtained from MBI Fermentas. Kits for plasmid isolation,

Chemicals

Log-phasecells grown in YPD medium containing or lacking CaCl2and FK506 were collected, PBS-washed and loaded with ratiometric, high affinity, membrane-permeable calcium indicator, Fura-2 AM (10 M; Sigma #47989). After 30 min incubation at 30◦C, labelled cells were washed thrice with cold PBS, suspended in PBS and fluorescence was recorded at 505 nm with dual excitation at 340 and 380 nm. The ratio of fluorescence intensities between 340 and 380 nm, representing Ca-bound and Ca-free Fura-2 molecules, respectively, reflected free intracellular calcium concentrations

Measurement of intracellular calciumlevels

the disrupted gene, BLAST N of the sequences from rescued plasmids was performedagainstC. glabrataGenolevures database (http://www.genolevures.org/blast.html

Identification of disrupted locus in Tn7insertion mutants was carried out as described previously(Kaur et al.,2004). Disrupted locus in each mutant is physically marked with a mini Tn7 transposon derivative containing conditional origin of replication R6K(facilitates Tn7recovery), S. cerevisiae URA3and Klebsiella pneumoniae hphgene (confers resistance to hygromycin B)(Castaño et al.,2003). Briefly,genomic DNA was isolated fromovernight grown Tn7insertion mutants using spheroplast lysis method. After RNAse treatment, 10μgDNA was either digested withMfe1or SpeIrestriction enzymeas the Tn7 cassette lacks these enzyme sites. Followingovernight digestion, DNA wasprecipitated with 1ml ethanol and 1/10thvolume of 3 M sodium acetate (pH 5.2).DNApellet was washed twicewith ice-cold 70% ethanol, air driedand resuspendedin sterile MQ water. DNA was recircularized withT4 DNA ligase. Resultant circular plasmid contains the Tn7cassette flanked on either side by the gene,it has disruptedin the genome of C. glabrata.This circular plasmid DNA was transformedin E.coliBW23473 strain,which contains protein Π (the product of the pir gene)required by R6Korifor replication.Transformation of circularized DNA in E.coliBW23473 electrocompetent cells was performedas described below.Plasmids fromselected transformants were isolated and sequenced with outward primers from Tn7right and left ends to sequencethe disrupted gene fragment.For identification of

Tn7insertion mutant rescueand gene identification

E. coli DH5α ultra-competent cells were transformed with plasmid DNA by heat shock at 42 ̊C for 90 sec as described previously in Molecular Cloning-A Laboratory Manual (Sambrook and Russell,2001). Bacterial transformants were selected on LB agarmediumcontaining appropriate antibiotics. Transformants obtainedwere colony purified on LB plates containing antibiotics.Presence of the desired insertwas first verified by colony PCR followed by PCRusing extracted plasmid DNA as template

Bacterial transformation

activated TLC silicagel-60plate and transferred to theTLC chamber. After the solvent had migratedupwards (1.5 cm fromthetop), TLC plate was removed, air dried behind perspex shield, wrapped with cling plastic wrap and was exposed to phophorimager screenfor 2 h. Phosphorimager screen was scanned usingaFugi-FLA 9000 scanner

To resolvephospholipids,a TLC chamber was prepared by pouring50 ml developing solution and sealing the chamber with aluminium foil so that developing solution can generate vapor. TLC silicagel-60plate(Merck)was incubated at 80ºCfor 4 h for activation. After 30 min of TLC chamber preparation, phospholipidsextracted from C. glabratacells werespotted at thebottom (1.5 cm fromthelowerend) of the

Seperation of phospholipids by thin layer chromatography(TLC)

PI-3kinase reaction was set up ina total volume of50μlin a 1.5 ml microcentrifuge tube as described below.PI-3 kinase reaction buffer = 25 μlSpheroplast lysate = 20 μl (equivalent to 10 μg protein)Sonicated phosphatidyl inositol = 5 μlReaction mix was incubated at 25ºC for 20 min and enzyme reaction was stopped by adding 80μlHCL (1N) solution. To extract phospholipids, 160 μl chloroform:methanol (1:1) was added to the reaction mix withcontinuous mixing. Organic phase containing phopholipidswas separated fromaqueous phase by centrifugation at 7,500g for 4 min at 4ºC and transferred to a new vial. Using vacuum evaporator apparatus, solvent was evaporated and phospholipidsweredissolved in 10 μl chloroform

PI-3 kinase reaction set up and phopsholipid extraction

10 mg phosphatidylinositol-sodium salt(from Glycine max)was dissolved in 2 ml chloroform to prepare a 5 mg/ml stock solution. This solution was prepared in a small glass vial aschloroformis known to reactwith polypropylene. Small aliquots of stock solution were madeand stored at -20ºC till further use. To avoid spillage due to vapor pressure, vials containing phosphatidylinositol-sodium salt solutionwereopened very carefully.To prepare sonicated phosphatidylinositolfor one PI-3 kinase reaction, 2 μlof the stock phosphatidylinositolsolution (10 μg) wastransferredtoanew1.5 ml microcentrifuge tube. Using vacuum evaporator apparatus, chloroformwas evaporated from the solution and phosphatidylinositol-sodium saltwas resuspended in 5 μl sonication buffer.For sonication, a total of 20 pulses, each of 30 sec with30 sec resting time weregiven on ice

Preparation and sonication of phosphatidylinositol-sodium salt solution

A single colonyof desired C. glabratastrainwas inoculated in YPD-liquid mediumand grown for 14-16 h. 50 μl overnight culture was inoculated inYPD-liquid mediumfor 4 h. Log-phase-grownyeast cells were harvested,washedwith PBSandwereinoculated atinitial OD600of 2 and 4,into YNB-dextrose and YNB-sodium acetate liquid medium,respectively.After 4 hincubation,yeast cells were harvested by centrifugation at 2,500g for 5 minand treated with 1.2 M zymolyasefor 1 hto obtain spheroplasts.Post zymolyase treatment, spheroplasts were resuspended in 100 μl resuspension bufferandanequal amount of 0.25 mm glass beadswasadded to lyse the spheroplasts. Using bead beater apparatus, spheroplasts were lysed and protein concentration in spheroplast lysateswas determined usingbicinchoninic acid assay (BCA) method and samples were stored at -20ºC till further use

Preparation of cell lysate

In vitroPI-3 kinase reactions wereset up to measure PI-3P synthesized as described earlier(Whitman et al., 1988)

Phosphatidyl inositol-3 kinase (PI-3 kinase assay)

C. glabrata cells were grown overnight in 10 ml YPD liquid medium. Cells were harvested at 2,500g for 5 min, resuspended in 400 μl Buffer A (50 mM Tris-HCl, 10 mM EDTA, 150 mM NaCl, 1% Triton X-100 and 1% SDS) and were transferred to a 2 ml microcentrifuge tube. Equal volume of phenol-chloroform-isoamyl alcohol (PCI) solution was added tocell suspension and tubes were vortexed for 2-3 min. After incubation at 42 ̊C for 30 minon a thermomixer set at 800 rpm (Eppendorf), cell debris was removed by centrifugation at 7,500g for 10 minand aqueous phase (300-350 μl) was carefully transferred to a new 2 ml microcentrifuge tube. Genomic DNA was precipitated with800 μl chilled absolute ethanol and 35 μl sodium acetate (3 M, pH 5.2). DNA pellet was washed with chilled 70% ethanol and dried at room temperature for 5-10 min. Genomic DNA pellet was dissolved either in 50 μl 0.1X TE or molecular biology grade water containing 0.3 μl Ambion RNase cocktail and incubated at 37 ̊C for 30 minfor digestionof RNA. After RNA degradation, 100 μl of 0.1X TE or nuclease-free water was added to the tube and stored at -20ºC. Quality of extracted genomic DNA was checkedon 0.6% agarosegel by electrophoresis

Genomic DNA isolationby quick genomic DNA extraction method

C. glabratastrains were grown overnight in YPD medium. Cellswereharvested from 1 mlcultureandwashed withPBS.Cells were next washed with50mM NaH2PO4andresuspendedin 100 μlFITC-dextran(50mg/ml). After incubation at 37°Cfor 45 min, cells were washed thrice with PBS for complete removal of FITC-dextran.Yeast cells were resuspended in 1 ml PBS and used to infect PMA-treated THP-1 cells in 4-chambered glass slide

Fluorescein isothiocyanate(FITC)staining of C. glabratacells

For confocal microscopyanalysis, 5X105THP-1 cells were seeded and treatedwithPMA in 4-chambered slides. Differentiated THP-1 macrophageswere infected either with FITC-labeled or GFP-expressingC.glabratastrains to a MOIof 1:1. At different time intervals, medium was aspirated out from each chamber of 4-chambered slides and chamberes were washed twice with PBS. To fixthe infected macrophages,500 μlformaldehyde(3.7%) was added gently toeach chamber andincubated for 15 minat room temperature. Each chamber of the slide was washed twice withPBS to remove formaldehyde solution completely. To permeabilize the fixed cells, 500 μl Triton-X (0.7%) was dispensed toeach chamber and slide wasincubated at room temperature for 5 min. Chambers of the slide werewashed twice with PBS, separated from the slideusing a chamber removal device andwere air dried. Coverslips were placed onslides using Vectashield mounting mediumand bordersweresealed withnail paint. Slides werestored at 4°C until used forfluorescence imaging

Fixing of PMA-treated THP-1 macrophages

Adherent cells growing either on cover slips or chamber slides were fixed with 4% paraformaldehyde for 10 min at room temperature. The cells were washed with PBS thrice for 5 min each and blocking was done in 2% BSA(preparedin PBScontaining 0.3% Triton-X 100) for 1h.The cells were incubatedwith primary antibody(dilutedin PBScontaining 0.3% Triton-X 100)for 2h at room temperature or overnight at 4⁰C.The cells were washed with PBS thrice for 5 min each followed by incubation withAlexa Fluor 488-or 594-conjugatedsecondary (anti-mouse/rabbit) antibodiesfor 1h. Then the cells were mounted on microscopicslides using Vectashieldmountingmediumcontaining nuclear dye DAPI. Imaging was done byeither the laser scanning confocal LSM510 or LSM 750 (Carl Zeiss, Oberkochen, Germany) or fluorescence inverted (Olympus 1X51, Tokyo, Japan) microscope

Immunofluorescence Microscopy

Neutralization solution(Solution III)

Lysissolution(Solution II)

Resuspension solution(Solution I)

For Plasmid isolation

Blocking Buffer

NP-40ComponentsFinal concentrationFor 10 mlNP-4010%1mlH2O9ml

Bradford method(Bradford, 1976)was used to determine the quantity of protein in various samplesin a 96-well plate. Bradford’s reagent was prepared by diluting Bradford dye with water in the ratio of 1:5.For estimating the concentration of protein in a particular sample, 50μl volume reactionwas set and200μl of freshly prepared Bradford’s reagent was added. The complex givesa purplish colorwhose intensity is proportional to the amount of protein present in the sample. A standard curve was also generated using increasing concentrations of BSA (50 μg/ml, 100 μg/mland 200μg/ml).Cell lysatesof test samples werediluted to1:50 in the same volume.Each sample (including blank and standards) was taken in duplicates.The concentration of protein was measuredusing the ELISA reader at 570 nm. The unknown protein concentration (X) was calculated as follows:where,OD1& OD2: Optical densities of Standard (Std) 1 & Standard (Std) 2, respectively.BSA: Bovine serum albuminX×50 (dilution factor)/1000 = YConcentration of unknown protein (μg/μl) = Y × OD

Estimationof protein concentration in cellular lysates

6XEMSA sample loading dye

5X EMSA buffer

Native EMSA PAGE

10XBinding buffer

For Electrophoretic Mobility Shift Assay (EMSA)

TBS-T

Transfer buffer

placed on ice.The samples were centrifuged at 10,000x gfor 10 min at 4°C and the supernatant was extracted by avoiding the glass beads. Absorbance of thelysatewas measuredat 260 nm, and it was divided into 200 μL aliquots and frozen at -80°C. No difference in the ribosome profiles were detected between thesesamples and fresh samples analysed immediately.Polysomes were analysed by centrifugation through a 10-50% sucrose continuous gradient. The gradient was prepared by pipetting, gradient buffers (Section 2.1.6.3), 5 mLof each layer onto the bottom of an 11 mLopen top centrifuge tube, covering the tube with paraffin filmand by placing it horizontally on a flat surface at 40C for 2 h. The ribosome sample (cell lysate) was loaded on top, and gradient was centrifuged at 100,000x gat4°C for 6 h in an SW41 rotor (Beckman). Anamount of lysate equivalent to 10 A260units was loaded on the gradient. Ribosome levels were measured by the gradient analysis with an ISCO UV-6gradient collector with continuous monitoring atA254.Polysomepurification was performed by layering the cell lysate on 37% sucrose solution in an 11 mLopen top centrifuge tube, and centrifuged at 100,000x gfor 14 h in an SW41 rotor(Beckman). The pellet was suspended in 50 to 100 μL of lysis buffer. An amount oflysate equivalent to 0.7A260unitswas loaded on the gradient. Ribosome levels were measured by gradient analysis with an ISCO UV-6gradient collector with continuous monitoring atA254.Ribosome subunits were analysedby centrifugation through a 10-30% sucrose continuous gradient. The gradient was prepared by pipetting 2.2 mLof each layer onto the bottom of a 5 mLopen top centrifuge tube, sealing it with paraffin filmand placing it horizontally on a flat surface at 4°C for 2 h.The ribosome sample wasloaded on top, and gradient was centrifuged at 100,000x gand 40C for 4.5 hin an SW55rotor (Beckman). Ribosome subunit levels were measured by gradient analysis with an ISCO UV-6gradient collector with continuous monitoring atA254.Ribosome profiles with yeast cell lysates and purification of ribosomes from yeast were performed with the help of Mr. Aluri Srinivasand Dr. Umesh Varshney

The method to analyse ribosome profiles was adapted from (Leeet al., 1992). Yeast cells were grown to 0.2 to 0.8 OD600at 30°C in 200mLof YPD,and cycloheximide was added to this media at 50μg/mLfinal concentration. The culture was placedand mixed continuously onanice and salt mixture for 2-5 min and centrifuged immediately at 4,000 x g. The culture was not allowed to stay for a longer time on the ice and salt mixture to avoid freezing. The cell pellet wassuspended in 1mLof lysisbuffer (Section 2.1.6.3),andtransferred to a2 mL microfuge tube, to which1mLglass beads (0.45-0.6mm diameter)were added, and lysed by bead beating for 10 min with intervals (30 sec on time and 1min off time).During the 1 min off time, tubes were

Ribosome profiles

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

lysed with ice-cold NETN lysis buffer (20mM Tris-HCl, pH 8.0, 100mM NaCl, 1mM EDTA, 0.5% Nonidet P-40) containing protease inhibitors (0.5 mM PMSF, 1 mg/ml aprotinin and 1 mg/ml pepstatin) for 30 min.on ice, and then centrifuged at 14000 RPM for 10 min. at 4°C. Protein concentration in the supernatant (cell lysate) was estimated by Bradford assay. 20ug of the protein lysate was mixed with6X SDS loading dye (100 mMTris pH6.8, 4% SDS, 0.2% bromophenol blue, 20% glycerol and 200 mMβ-mercaptoethanol), boiled for 5 minutes at 99°C. Protein samples were resolved electrophoreticallyon SDS-polyacrylamide gels and transferred onto PVDF-membrane (Amersham Biosciences) using a semi-dry transfer apparatus (Biorad) for 1h at a constant current of 500 mA. Membranes were blocked for 30 min.with 5% non-fat dry milk powderdissolvedin 1X PBS and then immunoblotted overnight with primary antibody diluted in blocking buffer. Membranes were washed four times (each 5 min.) with 1X TBST followed by incubation with secondary antibody conjugated with horseradish peroxidase (HRP) for one hour at room temperature. The membrane was washed four times with 1X TBST (each wash for 5 min.), and the specific proteins on the membrane were detected by using enhanced chemiluminescent (ECL) reagents in 1:1 ratio (Amersham)

At 24 or 48 h of transfection, cell culture media was removed, cells were collected in 1X PBS by scraping them off the plate. Cells were harvested by centrifugation at 4000 RPM for 5 min.at 4° C. Pellet was washed twice with ice-cold 1X PBS and

Western blotting or immunoblotting

For biofilm and attachment assays, Xanthomonas oryzaecells were grown in PS media with appropriate antibiotics at 28°C with constant shaking at 200 rpm. 0.2% of the overnight grown culture was inoculated into the fresh PS media and grown till the OD reached 0.6-0.7 at 600 nm. 4 ml culture was inoculated into 12 well polystyrene culture plates, and incubated for 24 h and 48 h at 28°C without shaking. After 24 h, cultures were discarded, and wells were washed with 4 ml of water to remove loosely attached cells. The adherence was examined by staining the cells with 1% crystal violet solution for 30 min at room temperature. After incubation, excess crystal violet stain was removed by washing the wells with 3 ml water. Images were captured for visualizing the stained biofilm on polystyrene plate. Finally, crystal violet stained biofilm was dissolved in 80% ethanol, and quantified by taking OD at 560 nm. Similar procedures were repeated for the polystyrene plate with culture incubated for 48 h. For attachment, cells were grown similarly in 12 well polystyrene culture plates for 24 h, rinsed once with sterile water to remove loosely attached cells then attached cells were collected by vigorous washing with sterile water. Attached cells were diluted, and plated to get the CFUs

Static biofilm and attachment assay

QIAGEN QIAquick Gel extraction kit containing required buffers, spin columns and collection tubes was used to extract and purify DNA from agarose gels. Digested DNA samples and PCR products were resolved on 1% agarose gel and gel piece containing desired fragment was cut on an UV-transilluminator. DNA fragment was purified following manufacturer’s instructions

Gel extraction of DNA

and finally resuspended in 100 μl sterile water. Bacterial cell suspension was aliquoted in 20 μl volume. The above procedure was followed for all the three strains and cell suspension of three different strains were mixed together in 1:1:1 ratio. For conjugation to occur, 20 μl of the above mixture was spottedon the LB agar plate and incubated at 37°C for 12-16 h. Next, the conjugation drops were streaked on LB agar plate containing appropriate antibiotics to select the S17-1 recipient containing recombinant plasmid.S17-1 was directly conjugated with Xanthomonasstrain. S17-1 strain containing recombinant plasmid (3 ml) and recipient Xanthomonasstrain (100 ml) was grown overnight with appropriate antibiotics. Cells were harvested and washed thrice as mentioned earlier. Xanthomonasstrain was finally dissolvedin 600-700 μl sterile water and S17-1 strain was dissolved in 3 ml sterile water. 50 μl Xanthomonascell suspension and 10 μl S17-1 cell suspension were mixed together and 20 μl was spotted on PS agar plate. After 40 h of incubation at 28°C, each conjugation drop was dissolved in 400 μl water separately and plated on PS agar medium with rifampicin (counter-selectable marker) and plasmid specific antibiotics for specific selection of Xanthomonascolony with recombinant plasmid

Since compatible conjugation does not exist between Xanthomonasand E.coliDH5α strain.Therefore, upon getting the appropriate clones in DH5α, conjugation was performed with S17-1 (recipient strain) and PRK600 (helper strain). All the three strains (DH5α with clone, S17-1 and PRK600 strain of E.coli) were grown overnight at 37°C with constant shaking at 200 rpm in 3 ml LB broth. Cells from 1 ml overnight grown cultures were harvested by centrifugation followed by three washes with s

Xanthomonas conjugation

To 1.0 ml of suitably diluted culture filtrate, 5.0 ml of solution C was added. It was incubated for 10 min at room temperature. To this, 0.5 ml of Folin Ciocalteau’s reagent (diluted 1:1 with distilled water) was added. The solution was vortexed and kept in dark for 30 min. After incubation, absorbance was read at 660 nm against a reagent blank. Protein content was calculated (in mg/ml) using standard curve of bovine serum albumin (BSA) prepared in the range 100-1000 μg/ml

The total protein content in the culture filtrate was estimated by Lowry’s method as described below: Reagents: Solution A: 2.0% Na2CO3 in 0.1 N NaOHSolution B: 0.5 % CuSO4 in 1.0 % Sodium potassium tartarate Solution C: 50.0 ml of solution A was mixed with 1.0 ml of solution B Folin Ciocalteau’s reagent

Protein estimation (Lowry et al., 1951)

To extract DNA from agarose gels,the QIAquick® gel extraction kit (QIAGEN, 28706) was used. For purification of PCR amplified DNA products,the QIAquick® PCR purification kit (QIAGEN, 28106) was used. Clean-up of enzymatic reactions was performedusing the MinElute® Reaction Cleanup kit (QIAGEN, 28204). Allprotocolswere followed as per manufacturer’s instruction

Gel extraction, PCR purification and Reaction clean-up

To extract DNA from agarose gels,the QIAquick® gel extraction kit (QIAGEN, 28706) was used. For purification of PCR amplified DNA products,the QIAquick® PCR purification kit (QIAGEN, 28106) was used. Clean-up of enzymatic reactions was performedusing the MinElute® Reaction Cleanup kit (QIAGEN, 28204). Allprotocolswere followed as per manufacturer’s instructions

Gel extraction, PCR purification and Reaction clean-up

The IP6K inhibitor TNP was dissolved in DMSO at a concentration of 22.6 mM and stored at -20°C. MEFs were seeded on coverslips in 12 well plates at 15% confluence. Once, cells adhered and attained their morphology, they were pre-treated with 5 μM TNP and DMSO 3 h prior to HU treatment. After 3 h, cells were treated with 0.5 mM HU for 12 h in presence or absence of TNP. Similarly, post drug removal,cells were incubated with or without TNP to monitorrecovery for 6 h. At each time point cells were processed for presence of nuclear BLM as described in Section 2.2.6. BLM was used as readout for initiation and completion of repair. Imageswere acquired as described in Section 2.2.6

Inhibition of IP6Ksin MEFs

Viewing slides under microscope

A drop of immersion oil was put on top of the cover-slip before viewing it under microscope. The cells were viewed at 100X resolution of Nikon Eclipse 80i microscope.Thedifferential interference contrast images of the cells were captured using NIS-Elements D3.0 software also used to find out mean cell size using at least 100 randomly selected cells.Fluorescence images were captured on Zeiss LSM 710 Meta inverted confocal microscope

The slides for microscopy were prepared as described in Dajkovic et al.,(2008)with slight modifications. After wiping the glass slide with ethanol, 200μL of 1% molten agarose was layered on it between two strips of tape and clean cover-slip placed on it to obtain levelled surface. The agarose was allowed to solidify and the cover-slip was carefully removed and5μlof sample was put on top of the agarose and carefully covered with a cover-slip

Preparation of microscopic slides

Fresh overnight cultures grown in LB containing appropriate antibiotics to select for plasmids were sub-cultured 1:100(or lower dilutions for some strains)in the same medium. The cells from these cultures weretaken for microscopy at exponential phase of growth(A600 of 0.5-0.6), as such or after concentrating the cells 10-fold

Sample preparation

Microscopy

Band intensities in gel autoradiogramswere determined by densitometry with the aid of the Fujifilm Multi Gauge V3.0 imaging system. Equal areas of radioactive bands were boxed and the PSL (Photo stimulated luminescence) values were further considered. Background signal (obtained from equal area as that of the radioactive band but from other part of the gel/blot) is subtracted from the signal intensities obtained from radioactive bands to get the final values

Densitometry

β-Galactosidase assay was performed according to(Miller, 1992).Cultures were grown to A600 of 0.4-0.6 from a 1:100 dilution of overnight cultures. Around 0.1-0.5 ml of culture was made up to 1 ml with Z-buffer and lysed with the addition of 100μl of chloroform and 50μl of 0.01% SDS solution. 0.2ml of freshly prepared 4mg/ml ONPG was added to start the reaction and incubated at 28oCtill the colour of the reaction mixture turned yellow. 0.5ml of 1M Na2CO3 was added to stop the reaction and the time duration from initial addition of ONPG to the stopping of the reaction was noted. The absorbance of reaction mix was taken at 420 nm (A420) afterspinning down the mix at 12000rpm for 3 minutes. The A600of the culturesused was also noted. The enzyme’sspecific activity (in Miller units) was calculated using the following equation: β-Galactosidase specific activity (Miller units) = (1000 ×A420) / t × v ×A600Where,‘t’ is the time period in minutes and ‘v’, the volume of culture used in ml

Chemicals were obtained from different commercial sources. Most of the chemicals such as amino acids, antibiotics, sugars, IPTG, ONPG and X-gal were obtained from Sigma Chemical Co. The media components for the growth of bacteria were mostly from Difco laboratories and Himedia. The materials used in the recombinant DNA experiments such as restriction endonucleases, T4-DNA ligase, DNA polymerases, polynucleotide kinaseand DNA size markers were obtained from companies including New England Biolabs, Invitrogen, Promega, Bangalore Genei, Sigmaand MBI Fermentas. Kits used for plasmids and genomic DNA isolation,purification of DNA fragments and PCR amplification were obtained from Qiagenand Invitrogen. High fidelity enzymes for PCR amplification were purchased from Sigma. The oligonucleotide primers used in this study were synthesized by Xcelris genomics orMWG Biotech. The radioactive chemicals were procured from Jonaki

Chemicals

ZFIN_ZDB-ALT-110818-7

ZFIN_ZDB-GENO-110912-7

They have encouraged and assisted thousands of our slaves to leave their homes; and those who remain, have been incited by emissaries, books and pictures to servile insurrection.

most commonly creep into the decision-making process.

one of the very strongest arguments in favor of the Confederation of the provinces, that it enables us to prepare appropriate defences along the whole frontier of our country.

We should, probably, in time aspire to have foreign relations of our own, to have our own army and navy, and to seek for that complete emancipation which with communities as with individuals, maturity prompts. But independence in a state must always be relative, and none of us can expect to live to see the day when the British dominions in this part of the world will be peopled to such an extent, and become so powerful, that they can afford to be independent of England. We must, from the necessities of our geographical position—so long as the United States continue to be as powerful as they are ; and even if they were divided into two or three portions—we must always find in them a source of danger which must force upon us a dependence on England.

Why, while there is yet time, should we neglect to take those salutary precautions on which our existence as French-Canadians depend ? Our Local Government ought to have the same active part in the organization, instruction and equipment of our militia which belongs to all local governments which form part of other confederacies.

the 67th resolution. I find by this resolution ” that the General Government will fulfil all engagements entered into, previous to the union, with the Imperial Government, for the defence of the country.” Now strange to say, the authors of this document do not even take the trouble to state by whom such engagements must be made. No, they simply assert the obligation in the terms of the resolution I have just quoted. Suppose our Government had entered into an engagement to the extent of fifty millions of dollars, shall we—can we—affirm that the engagement was a necessary one, by voting for the measure without knowing the nature of the engagement ?

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

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

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.

As part of the joint project "Innovation Network Morgenstadt: City Insights" under the project management of the Fraunhofer-Gesellschaft for the Promotion of Applied Research eV, the ESRI (Environmental Systems Research Institute) is developing a three-dimensional visualization of the district of Mülheim in cooperation with the City of Cologne. For this purpose, the city of Cologne provides data from the areas of environment, traffic, real estate, urban planning. From this three-dimensional geoinformation system (so-called 3-D GIS model), an app is being developed, which is expected to be available to the people of Cologne in the second quarter of 2015 on the homepage of the city of Cologne.

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.  

n times of energy transition and scarce resources, the architectural concept of Concrete Apartments Cologne is based on the requirements of the future - it is designed as an energy-saving passive house. This contains • a 26 cm thick external insulation made of rock wool, • triple glazed windows, • optimum recovery of radiated heat from residents and household appliances, • a ventilation system with a constant base temperature of 20 ° C - summer and winter - as well as • a digital control system that directs the use of luminaires and large consumers. Only those who like it even warmer must turn on the heating controller. All rooms are equipped with presence detectors, which automatically switch off lamps, for example, when not in use - this also saves energy. Of course, residents can also make the scheme manually. The energy and heat for the Boarding House creates its own, energy-efficient combined heat and power plant. State-of-the-art technology is also used here: surplus electricity is optionally fed into the public grid or used for the charging station for electric vehicles in the courtyard.

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.

nebenan.de is a free, local platform for building and maintaining neighborly relationships. Get to know, share, help, give, inform, get together - nebenan.de offers neighbors the opportunity to get in touch and to live the neighborhood actively. nebenan.de is with more than 650,000 active users in currently around 5,500 neighborhoods Germany's largest social network for building and maintaining neighborly relations. Any resident can join his neighborhood on nebenan.de or initiate it himself.   nebenan.de offers the comprehensive solution for simplifying and revitalizing neighborly exchanges via the browser or as Android and iOS app.

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.

The energy transition presents network operators and energy providers with particular challenges. Both have to deal with an increasing share of electricity from renewable sources in the electricity grid. Wind turbines and photovoltaic systems produce electricity, however, depending on the weather and therefore fluctuating. For a secure supply, it is necessary that electricity production and consumption are always balanced as much as possible. In order for this to succeed, utilities and network operators must always know where and in what quantity energy is generated and consumed. Only then can production and consumption be optimally coordinated. However, continuous metering is not possible with today's metering technology.The solution to the problem is smart metering. In the future, so-called "smart metering systems" will transfer consumption data to grid operators and energy providers. This ensures that they can optimally control the network at any time. The technology is mainly used in households and businesses with high annual consumption.Consumers can access the data at any time. The additional transparency helps them to further increase their energy efficiency and thereby reduce costs. New services provided by energy suppliers are intended to reinforce the positive effects.

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.

This I contend, then, that if the military and naval defences of all the provinces are to be provided for by the General Government, and if you have to increase the militia for this purpose, the Lower Provinces will pay only their proportion of two-twelfths, and Canada, while obtaining no greater defensive force than at present, will have to pay five times as much as we are now paying.

HamburgService - Kita-Infosystem

District heating is one of the key pillars of our sustainable energy action plan. This plan has been decided by the local parliament in 2008 and renewed in 2015. Our first priority is to cut in half the total energy demand of the city until 2050 and then cover the rest with renewable energy and/or waste heat. To use large amounts of waste heat (e.g. from a waste incineration plant, industry, datacentres …) you need a distribution system, because it is not useable only locally. This is why we want to increase the share of district heating in the city. For the future we see a district heating system which will be “open source technology” – everyone can use the heat and also be a prosumer, delivering surplus energy, e.g. from a solar – thermal plant, to the system. There will not be any longer central DH-Stations but smaller plants and the use of all waste heat sources we can get.

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!

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

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!

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

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

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

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

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).

Hamburg Open Online University Los geht's!

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.

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.

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.

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.

Nav­i­ga­tion in real-time  Thou­san­ds of trucks drive through the port of Ham­bur­g every day. To en­sure that the traf­fic flows ef­fici­ently, the HPA com­bines var­i­ous ser­vices and func­ti­ons. Any­one dri­ving around the port ben­e­fits from per­so­na­li­sed nav­i­ga­tion. As well as in­for­ma­tio­n about the traf­fic sit­u­a­tion in and around the port, they also have ac­cess to park­ing and in­fra­struc­ture ­in­for­ma­tio­n, clo­sures of the move­able bridges, as well as the lat­est in­for­ma­tion on im­por­tant op­er­a­tions.

smart­PORT lo­gis­tics Thanks to in­tel­li­gen­t so­lu­tions for the flow of traf­fic and goods, the HPA is im­prov­ing the port's ef­fici­ency. smart­PORT lo­gis­tics com­bines eco­no­mic and eco­lo­gical as­pec­ts in three sub-sec­tors: traf­fic flows, in­fra­structure and the flow of goods. An in­ter­mo­da­l Port­Traf­fic cen­tre for sea, rail and road trans­port forms the ba­sis for net­work­ing the flow of traf­fic. In­tel­li­gen­t net­work­ing is a pre­req­ui­site for smooth, ef­fici­en­t trans­port in the port of Ham­bur­g and ul­ti­mately for the flow of goods: op­ti­mum da­ta cap­ture and rapid in­for­ma­ti­on shar­ing al­low lo­gis­tics man­agers, car­ri­ers and agen­ts to se­lect the most efficien­t means of trans­port for their goods.

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

Discover cashless, ticketless and hassle-free on-street parking with ParkNow in Berlin. The digital parking service ParkNow can be used in the extended inner city area of Berlin. A detailed overview of all parking zones can be obtained from our overview map. In the marked parking zones you can start and end your parking via app and save yourself the annoying way to the parking meter as well as the search for change. No more unnecessary parking fees thanks to minute-accurate billing and start / stop function. The billing occures convenient at the end of the month via direct debit, PayPal or credit card. The advantages of mobile parking with ParkNow in Berlin: Parking tickets are a thing of the past No more searching for small change and ticketing problems Accurate billing Comfortable payment at the end of the month Parking in Berlin was never that easy

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.

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.

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.

Innovationsassistent/-in - Innovation assistant New know-how for your company Are you in the start-up phase and are looking to position your company successfully on the market? Or is your company already established and you are now looking to expand? One way to achieve this is to hire a qualified graduate from a university or a university of applied sciences. IBB works on behalf of Berlin's Senate Department for Economics, Technology and Research in order to provide funding for hiring innovation assistants.

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.

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.

What do you want to do? Find services, locations authorities, opening hours and much more!

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.

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