For Immunoblotting

Sodium Chloride (NaCl)ComponentsFinal concentrationFor 100 mlNaCl5M29.22gH2Oq.s

Permeabilisation buffer: 0.2% Triton X100

(c) 6X Protein loading buffer (Lammeli buffer)

developer solution for appropriate time and immediately kept in fixer solution to see the protein band. For alkaline phosphatase method, blot was incubated with 5 ml of BCIP/NBT solution (Amresco) under dark condition. After incubation, blot was washed with water to see the blue-violet color protein band

volume of 50 mM acetate buffer (pH-5.4), and dialyzed overnight with 10 mM Tris buffer, pH 7.5. Pellet was used for dilution plating for calculating CFUs. For whole cell protein isolation, bacterial pellet was dissolved in 50 mM sodium acetate buffer (pH-5.4) and sonicated for 30 min (1 min on and off, Amplitude 32) by adding phenylmethylsulfonyl fluoride (PMSF) at a final concentration of 1 mM in ice-cold solution. Both extracellular proteins and whole cell lysate fractions were aliquoted in 1.5 ml microcentrifuge tube, and protein quantification was performed using a Pierce BCA protein assay kit (Thermo Scientific) as per manufacturer’s instructions using bovine serum albumin as standard and stored at -80°C for further use. Cell normalized extracellular and whole cell lysate proteins fractions from different strains were resolved on 12% SDS-PAGE gel at 90 V till the dye front reached the bottom. One gel was processed for silver staining (Sambrook et al., 1989), and other for western-blot analysis by using anti-GFP antibody. For western blot analysis, resolved proteins were transferred to Hybond-ECL membrane (Amersham biosciences) at 35 V for overnight in the cold room. Transfer of the proteins were visually confirmed by examining marker’s lane and membranes were incubated in small box for 2-3 h in 5% fat free milk prepared in 1X PBST for blocking. Blocking solutions were discarded, and primary antibody, appropriately diluted in 5% fat free milk prepared in 1X PBST, was added to the box containing membrane. After 2-3 h incubation in primary antibody, membranes were washed thrice with 1X PBST for 10 min. Membranes were incubated for 2 h in appropriate secondary antibody (anti-Rabbit antibody)diluted in 5% fat free milk prepared in 1X PBST. Blots were either developed by chemiluminescence based ECL-plus western detection system or alkaline phosphatase method. For HRP based chemiluminescence method, detection was performed using the ECL plus kit (Amersham biosciences) and incubated for 3 min. Blot was exposed to the film and developed i

For protein extraction, Xanthomonas oryzaepv. oryzaestrains with eGFP plasmid were grown for 24-30 h in PS medium to an OD of 0.8 as described above and centrifuged at 12,000 g for 10 min. The supernatant was taken as extracellular fraction and protein was extracted as described previously (Ray et al., 2000). Extracellular proteins were precipitated from this fraction by constantly adding 50% (wt/vol) ammonium sulphate at 4°C. After precipitation, the solution was kept on ice for 15-20 min and centrifuged at 12,000 g for 30 min at 4°C. The pellet was dissolved in s

Protein extraction and immunoblotting

work were autoclaved twice and dried at 80°C for overnight before use. RNA was isolated from Xanthomonasculture using Trizol method. Xanthomonascells were harvested at 12,000 g for 5 min at 4°C, resuspended in approximately 1 ml Trizol (Invitrogen),mixed properly and incubated at room temperature (RT) for 5 min. 200 μl chloroform was added to the tube, shaken for 15 seconds and incubated at RT for 2-15 seconds. Next, tubes were centrifuged at 13,000 g for 15 min at 4°C. Aqueous phase was transferred to new 1.5 ml microcentrifuge tube and RNA was precipitated by adding 500 μl isopropanol and incubated for 5-10 min at RT. Precipitated RNA was collected by centrifugation at 10,000 gfor 10 min at 4°C. RNA pellet was washed with 70% ethanol and resuspended in 20 μl nuclease-free water. RNA concentration was determined by measuring absorbance at 260 nm. Quality of RNA was examined by gel electrophoresis on 0.8% agarose gel with TAE buffer prepared in DEPC treated water

For RNA experiments, all solutions were prepared in RNase free diethylpyrocarbonate (DEPC) treated water. Microcentrifuge and tips u

RNA extraction

microcentrifuge tube. For precipitation of RNA, 1/10thvolume of 3 M sodium acetate (pH 5.3) and 2.5 volume of 100% ice-coldethanol was added. In order to facilitate precipitation, tubes werekept at -20°C for 20 min. Tubes were centrifuged at 13,000 rpm for 10 min in a refrigerated centrifuge. The RNA pellet was washed with 70% ethanol,resuspendedin 100-200 μl of nuclease-free water and stored at -20°C untiluse.Care was taken to keep allreagents and tubes on ice to maintain the cold temperature throughout theRNA extractionprocess

All reagents required for RNA extraction were preparedin DEPC-treated water. RNasecontamination from non-autoclavable items wasremoved by wiping them with RNaseZap® (Ambion). Total RNA from yeast cells was extractedusing acid phenolextractionmethod. Briefly, yeast cells were grown underappropriate conditions and at suitabletime points,cells were harvested by centrifugation at 4,000 rpm for 5 min. The cell pellet was washed twice with ice-cold DEPC-treated water, resuspended in 350 μl of AE buffer and transferred toa1.5 ml microcentrifuge tube. To this,40 μl of 10% SDS and 400 μl of acid phenol (pH 4.3) was added. The cell suspension was mixed well by vortexing thrice, short pulsesof10 seconds each,and incubated at 65°C for 15 min with continuous agitation at 800 rpm. Post incubation, cells were kepton ice for 5 min and centrifuged at 13,000 rpm in a refrigerated centrifuge set at 4°C for 10 min. After centrifugation, aqueous layer was transferred to a new1.5 ml microcentrifuge tube and 400 μl of chloroform was added. Tubes were mixed well by gentlyinverting them 4-5 times and centrifuged at 13,000 rpm for 10 min. The aqueous layer was separated and transferred to a new1.5 ml

Total RNA isolation

This method was used to isolate highly pure genomic DNA. Briefly, 10 ml overnight grownC. glabratacultures were spun downandwashed with 10 ml sterile water. Washed cells wereresuspended in500 μl sterile water and transferred toa1.5 ml microcentrifuge tube. Tubes were spundownat 4,000 rpm for 5 min, supernatant was discarded andcell pellet was resuspended in 500 μl of buffer containing 100 mM EDTA and 5% β-mercaptoethanol and incubatedat 42°C for 10 min. Post incubation, cells were spun down at 4,000 rpm for 5 min and resuspended in freshly prepared Buffer B. To this, one tip-full of lyticase (Sigma, L4025) was added and incubated at 37°C for 1 h.After incubation, spheroplasts were collected by spinning downtubes at 6,000 rpm for 5 min, supernatant was discarded and the pellet was resuspended in 500 μl of Buffer C. DNA was extracted twice with 500 μl of PCI (25:24:1) solution and the aqueous layer was transferred toa new1.5 ml microcentrifuge tube. To this, 2.5 volume of absolute ethanol and 1/10thvolume of 3 M sodium acetate (pH 5.3) wereadded. Tubes were spundownat 13,000 rpm for 10 min, DNA pellet was resuspended in 200 μl of 1X TE buffer containing0.3 μl of RNase cocktail (Ambion) and incubated at 37°C for30 min. DNA was precipitated again by adding absolute ethanol and sodium acetate as mentioned above. DNA pellet was washed once with 70% ethanol, centrifuged at 13,000 rpm for 10 min, air-dried at room temperature and was resuspended in 100-200 μl of 1X TE buffer by gently tapping the tube. DNAwas stored at -20°C until use

Spheroplast lysis method

microcentrifuge tube. For precipitation of RNA, 1/10thvolume of 3 M sodium acetate (pH 5.3) and 2.5 volume of 100% ice-coldethanol was added. In order to facilitate precipitation, tubes werekept at -20°C for 20 min. Tubes were centrifuged at 13,000 rpm for 10 min in a refrigerated centrifuge. The RNA pellet was washed with 70% ethanol,resuspendedin 100-200 μl of nuclease-free water and stored at -20°C untiluse.Care was taken to keep allreagents and tubes on ice to maintain the cold temperature throughout theRNA extractionprocess

All reagents required for RNA extraction were preparedin DEPC-treated water. RNasecontamination from non-autoclavable items wasremoved by wiping them with RNaseZap® (Ambion). Total RNA from yeast cells was extractedusing acid phenolextractionmethod. Briefly, yeast cells were grown underappropriate conditions and at suitabletime points,cells were harvested by centrifugation at 4,000 rpm for 5 min. The cell pellet was washed twice with ice-cold DEPC-treated water, resuspended in 350 μl of AE buffer and transferred toa1.5 ml microcentrifuge tube. To this,40 μl of 10% SDS and 400 μl of acid phenol (pH 4.3) was added. The cell suspension was mixed well by vortexing thrice, short pulsesof10 seconds each,and incubated at 65°C for 15 min with continuous agitation at 800 rpm. Post incubation, cells were kepton ice for 5 min and centrifuged at 13,000 rpm in a refrigerated centrifuge set at 4°C for 10 min. After centrifugation, aqueous layer was transferred to a new1.5 ml microcentrifuge tube and 400 μl of chloroform was added. Tubes were mixed well by gentlyinverting them 4-5 times and centrifuged at 13,000 rpm for 10 min. The aqueous layer was separated and transferred to a new1.5 ml

Total RNA isolation

This method was used to isolate highly pure genomic DNA. Briefly, 10 ml overnight grownC. glabratacultures were spun downandwashed with 10 ml sterile water. Washed cells wereresuspended in500 μl sterile water and transferred toa1.5 ml microcentrifuge tube. Tubes were spundownat 4,000 rpm for 5 min, supernatant was discarded andcell pellet was resuspended in 500 μl of buffer containing 100 mM EDTA and 5% β-mercaptoethanol and incubatedat 42°C for 10 min. Post incubation, cells were spun down at 4,000 rpm for 5 min and resuspended in freshly prepared Buffer B. To this, one tip-full of lyticase (Sigma, L4025) was added and incubated at 37°C for 1 h.After incubation, spheroplasts were collected by spinning downtubes at 6,000 rpm for 5 min, supernatant was discarded and the pellet was resuspended in 500 μl of Buffer C. DNA was extracted twice with 500 μl of PCI (25:24:1) solution and the aqueous layer was transferred toa new1.5 ml microcentrifuge tube. To this, 2.5 volume of absolute ethanol and 1/10thvolume of 3 M sodium acetate (pH 5.3) wereadded. Tubes were spundownat 13,000 rpm for 10 min, DNA pellet was resuspended in 200 μl of 1X TE buffer containing0.3 μl of RNase cocktail (Ambion) and incubated at 37°C for30 min. DNA was precipitated again by adding absolute ethanol and sodium acetate as mentioned above. DNA pellet was washed once with 70% ethanol, centrifuged at 13,000 rpm for 10 min, air-dried at room temperature and was resuspended in 100-200 μl of 1X TE buffer by gently tapping the tube. DNAwas stored at -20°C until use

Spheroplast lysis method

To phenotypically characterize C. glabratamutants,serial dilution spot growth assays were performed. Briefly, the optical density of overnight-grown C. glabratacultures wasnormalized to OD600of 1.0andnormalized cultures were further diluted 10-fold in 1X sterile PBS five times. 3 μl of serially diluted culture were spotted on test plates. Plates were incubated at 30°C (unless mentioned otherwise) for 24-48hand growth was recorded by capturing plate images. For experiments involvingchecking theability of mutants to utilize non-fermentable carbon sources,growth was scoredafter 6-7 days of incubation

Serial dilution spot growth assay

Total RNA extraction from E. colicells was doneusing Qiagen RNeasy minikit. Cells were grown to an A600of 0.6 and harvested(amaximumof107cells)at 6000rpm for 5min at room temperature to prevent cells for encountering any stress in cold. Rest of the steps were followed exactly as mentioned in the manufacturer’s protocol. The quality of RNA preparations was assessed following electrophoresison 1.4% agarose-formaldehyde-MOPS gels.Ingeneral,forawild-typestrainRNAyieldwouldbe~0.5-1μg

Isolation of total cellular RNA

Restriction enzyme digestion and analysis

accustom the people to direct taxation

however, have said that they were in favor of direct taxation for the support of the local governments, because it would lead those who have to pay the taxes to look more closely into what was going on, and the manner in which their money was expended. (Hear, hear.) There seems also to have been a feeling in the Lower Provinces in favor of a legislative union, and the Hon. Mr. GREY seems to be combatting that idea. He says that with a legislative union, municipal institutions, and direct taxation in every province, would be the only means of getting along. He expressed himself as opposed to that and in favor of a Federal union, which he thought would afford them all the advantage that could be attained, commercially, by union, and would allow each province to retain control over its own local affairs. The local legislatures, he said, were to be deprived of no power over their own affairs that they formerly possessed. But in Canada it was represented that the local legislatures were to be only the shadow of the General Legislature—that they were to have merely a shadow of power, as all their proceedings were to be controlled by the Federal Government. That is the position taken by the advocates of the measure on this floor. So it seems that those gentlemen who have represented to us that they acted in great harmony, and came to a common decision when they were in conference, take a widely different view of the questions supposed to have been agreed upon, and give very different accounts of what were the views of parties to the conference on the various subjects. (Hear, hear.) In the Lower Provinces they were strongly opposed to direct taxation, while here it was present end as one of the advantages to accrue from the Federation. (Cries of No, no.) Well, Mr. SPEAKER, I say yes. That view of the case has been taken. If the amount allowed for the expenses of local legislation—the 80 cents per head—was found insufficient, the local parliaments must resort to direct taxation to make up the deficiency, while in tile Lower Provinces, it seems, nothing of that kind was to follow.

Another question on which the hon. member has also called us to account, relates to the export duties on timber and coals. In clause 29, which relates to the powers of the Federal Parliament, the third section reads as follows : This imposition or regulation of duties of customs on imports or exports, except on exports of timber, logs, masts, spars, deals, and sawn lumber from New Brunswick, and of coal and other minerals from Nova Scotia. The fact that this power has been conferred on the Government does not imply that it will be exercised. The power was granted simply because it might be necessary in certain cases mentioned. Now this is the reason for the second part of the clause which I have just read to the House, and which I cannot better explain than by citing some expressions of a speech by the Hon. the Minister of Finance on the subject. Nevertheless, as there are several honorable members in the House who do not understand English, I think it will perhaps be better to explain them in French. Here then was the thought of the Convention : as in New Brunswick the Government had found that it was a great disadvantage to collect the duties on timber according to the system formerly adopted, and they had substituted an export duty which superseded all other dues on that product, it was no more than right that this source of revenue should remain in New Brunswick, to which province it was an object of absolute necessity to defray its local expenses. In Canada we retain, under the new Constitution, our own method of collecting similar duties. As to New Brunswick, the duty on the article in question is their principal revenue, as coal is almost the sole revenue of Nova Scotia ; and if they had been deprived of them, they would have peremptorily refused to join the Confederation. (Hear, hear.) Their demand was perfectly just, and could not therefore be refused. Moreover, we have no right to complain, for they leave us all our mines and our lands, and we shall now, as heretofore, collect the proceeds for our own use and profit. The honorable member for Hochelaga says that it will be impossible to administer the affairs of the local legislatures without having recourse to direct taxation ; but a man of his experience ought not to have made that assertion. Instead of attempting to trade on popular prejudice, he ought to have admitted at once that the right granted by the new Constitution of levying direct taxes, is the same that already exists in the present Constitution ; it is the same right that all our municipalities possess.

reference to the meaning of the 5th sub-section of the 29th clause, which commits to the General Parliament ” the raising of money by all or any other modes or system of taxation.” Am I to understand that he General Government are to have the power of imposing local taxation upon the lands of the provinces ? HON. MR. CAMPBELL—The general national power of taxation is to be in the General Government.

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.

the Central Government would have the power of raising money by all the other modes and systems of taxation—the power of taxation had been confided to the General Legislature—and there was only one method left to the Local Governments, if their own resources became exhausted, and this was direct taxation.

Cindy A. Buckmaster: Animal research Is a labor of love for animals and people

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and Frank R.

Chapter 1, The Art of Community We begin the book with a bird’s-eye view of how communities function at a social science level. We cover the underlying nuts and bolts of how people form communities, what keeps them involved, and the basis and opportunities behind these interactions. Chapter 2, Planning Your Community Next we carve out and document a blueprint and strategy for your community and its future growth. Part of this strategy includes the target objectives and goals and how the community can be structured to achieve them. PREFACE xix Chapter 3, Communicating Clearly At the heart of community is communication, and great communicators can have a tremendously positive impact. Here we lay down the communications backbone and the best practices associated with using it