strain: PAO1 mode of growth: Biofilm calcium level: 10 mM
Treatment protocol
Cells were cultured in BMM with no added calcium, or with 10 mM added calcium.
Growth protocol
planktonic_samples: Cells were cultured planktonically for 18 hours in BMM at 37°C with shaking, as described in detail in the related publication. biofilm_samples: Cells were also cultured in biofilms on the interior surface of silicone tubing for 72 hours at 37°C with BMM flowing at 2 mls per minute, as described in detail in the related publication.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using a hot phenol method. Pelleted cells were suspended in 1.5 mls lysis buffer (0.15 M sucrose, 0.01 M sodium acetate, pH 4.5) and 1.5 mls 2% sodium dodecyl sulfate. Following the addition of 3 mls of water-saturated phenol, the mixture was incubated for 5 minutes at 65°C with frequent vortexing. 3 mls of chloroform were added, and the mixture was centrifuged for 30 minutes at 4°C. The aqueous phase was precipitated overnight, washed, and resuspended in RNase-free water. The RNA was cleaned on an RNeasy column (Qiagen) following the manufacturer’s mini cleanup protocol and then subjected to two 30 minute Turbo DNase (Ambion) treatments before precipitation and resuspension in RNase free water.
Label
Cy3
Label protocol
8 micrograms of total RNA were primed with 1 ul random hexamers (Invitrogen) during a 10 minute 70°C incubation in the presence of 1 µl RNase-in plus (Promega). After a thirty second incubation on ice, RNA was then reverse transcribed at 42°C overnight using the following reagents from Invitrogen: 5.5 µl 5x first strand buffer, 3 µl 0.1 M DTT, 0.6 µl 25mM modified dNTP mix (2:1 ratio of amino-allyl dUTP to dTTP), and 2 µl Superscript II. After synthesis, the remaining RNA was hydrolyzed by the addition of 0.5 M EDTA and 1 N sodium hydroxide. Next, resulting cDNA samples were neutralized with 1 N HCl and 1 M sodium acetate and cleaned on MinElute columns (Qiagen) according to the manufacturer’s instructions with the substitution of non-tris containing buffers as specified in SOP#M007 (JCVI). The resulting cDNA was assessed for quantity and purity using the Nanodrop1000 (Thermo Scientific). It was then dried to completion with 40°C heat in an RC10-10 speed vac (Jouan) and resuspended in 9.0 µl sodium carbonate buffer, pH 9.0. Each cDNA sample was divided into two equal portions and labeled with either Cy 3 or Cy 5 dye (GE Healthcare), again according to SOP#M007 (JCVI), for 90 minutes. Unincorporated dye molecules were removed with the Qiagen MinElute clean-up kit and labeled samples were eluted with 25 µl EB buffer. Corresponding samples co-hybridized to the same array were combined into a 0.5 ml centrifuge tube.
strain: PAO1 mode of growth: Biofilm calcium level: 0 mM
Treatment protocol
Cells were cultured in BMM with no added calcium, or with 10 mM added calcium.
Growth protocol
planktonic_samples: Cells were cultured planktonically for 18 hours in BMM at 37°C with shaking, as described in detail in the related publication. biofilm_samples: Cells were also cultured in biofilms on the interior surface of silicone tubing for 72 hours at 37°C with BMM flowing at 2 mls per minute, as described in detail in the related publication.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using a hot phenol method. Pelleted cells were suspended in 1.5 mls lysis buffer (0.15 M sucrose, 0.01 M sodium acetate, pH 4.5) and 1.5 mls 2% sodium dodecyl sulfate. Following the addition of 3 mls of water-saturated phenol, the mixture was incubated for 5 minutes at 65°C with frequent vortexing. 3 mls of chloroform were added, and the mixture was centrifuged for 30 minutes at 4°C. The aqueous phase was precipitated overnight, washed, and resuspended in RNase-free water. The RNA was cleaned on an RNeasy column (Qiagen) following the manufacturer’s mini cleanup protocol and then subjected to two 30 minute Turbo DNase (Ambion) treatments before precipitation and resuspension in RNase free water.
Label
Cy5
Label protocol
8 micrograms of total RNA were primed with 1 ul random hexamers (Invitrogen) during a 10 minute 70°C incubation in the presence of 1 µl RNase-in plus (Promega). After a thirty second incubation on ice, RNA was then reverse transcribed at 42°C overnight using the following reagents from Invitrogen: 5.5 µl 5x first strand buffer, 3 µl 0.1 M DTT, 0.6 µl 25mM modified dNTP mix (2:1 ratio of amino-allyl dUTP to dTTP), and 2 µl Superscript II. After synthesis, the remaining RNA was hydrolyzed by the addition of 0.5 M EDTA and 1 N sodium hydroxide. Next, resulting cDNA samples were neutralized with 1 N HCl and 1 M sodium acetate and cleaned on MinElute columns (Qiagen) according to the manufacturer’s instructions with the substitution of non-tris containing buffers as specified in SOP#M007 (JCVI). The resulting cDNA was assessed for quantity and purity using the Nanodrop1000 (Thermo Scientific). It was then dried to completion with 40°C heat in an RC10-10 speed vac (Jouan) and resuspended in 9.0 µl sodium carbonate buffer, pH 9.0. Each cDNA sample was divided into two equal portions and labeled with either Cy 3 or Cy 5 dye (GE Healthcare), again according to SOP#M007 (JCVI), for 90 minutes. Unincorporated dye molecules were removed with the Qiagen MinElute clean-up kit and labeled samples were eluted with 25 µl EB buffer. Corresponding samples co-hybridized to the same array were combined into a 0.5 ml centrifuge tube.
Hybridization protocol
All hybridization steps were carried out with slight modifications to SOP#M008 (TIGR). Briefly, slides were soaked at 42°C in 50 mls prehybridization solution (5x SSC, 0.1% SDS, 1% BSA) for a minimum of 60 minutes to block non-specific hybridization. Slides were then transferred to a falcon tube containing 50 mls of filtered, autoclaved deionized (FAD) water, which was shaken by hand for 30 seconds. This was repeated a minimum of three times before the slides were transferred to 50 mls isopropyl alcohol and shaken at room temperature for two minutes. Slides were then dried by centrifugation for 30 seconds in a slide spinner and visually confirmed to be free from streaking and all residue. A master mix of hybridization solution containing 50 µl formamide, 25 µl 20X SSC, 5 µl 2% SDS, 1 µl DTT and 19 ul FAD water was prepared. To each combined, labeled cDNA probe (which consists of Cy 5 labeled test RNA and Cy 3 labeled reference RNA, and vice versa), 35 µl of hybridization mixture, 1 µl tRNA, and 1 µl of herring sperm DNA was added. This mixture was denatured at 95°C for three minutes and allowed to cool slightly before being slowly injected by pipette onto the surface of the slide array under a carefully positioned and cleaned LifterSlip™ (Erie Scientific). The slide was then placed in a prewarmed hybridization chamber (Corning, cat # 2551), sealed, wrapped with aluminum foil, and submerged in a 43°C water bath for 16 to 20 hours. All subsequent steps were performed while keeping the slide protected from light. Following hybridization, LifterSlips™ were removed from the slide by gentle immersion in 43°C buffer containing 2X SSC, 0.2% SDS, and 0.01 mM DTT. Next, slides were placed in this 43°C low stringency buffer and shaken for four minutes. Slides were then transferred to room temperature medium stringency buffer containing 1x SSC, 0.1% SDS, and 0.01 mM DTT and shaken for four minutes. This was repeated before slides were again transferred to room temperature high stringency (0.1x SSC, 0.01 mM DTT) buffer and shaken for four minutes. This high stringency wash was repeated twice for two minutes each before slides were dipped in FAD/0.01 mM DTT water twice, dried in a slide spinner for one minute, and immediately scanned.
Scan protocol
Slides were scanned using the GenePix 4000B scanner (Molecular Devices) and emissions at 532 and 650 nm were recorded. Genepix Pro V 6.0 software (Molecular Devices) was used to obtain median pixel intensity values for each spot on the array and generate GenePix Results (gpr) files.
Description
PAO1 biofilms at 0 and 10 mM calcium, biological replicate 2 dye swap
Data processing
Gpr files were imported into Flexarray v 1.6.1 for normalization and analysis. Background correction was performed using the normexp algorithm. The Loess algorithm was used for within-array normalization, followed by scaling for between-array normalization. The Limma TREAT (t-tests relative to a threshold) method was used to generate symmetrical fold change to conservatively assess differential expression due to calcium addition. Empty and reserved spots were not included in the dataset. Replicate spots were averaged and genes with greater than two-fold change at p< 0.05 were selected for further analysis.
The response of Pseudomonas aeruginosa strains PAO1 and FRD1 to 10 mM calcium under planktonic and biofilm conditions
Data table header descriptions
ID_REF
VALUE
Fold change of 10 mM calcium over 0 mM calcium; ratio of background subtracted, loess normalized, scaled median pixel intensity of 10 mM / 0 mM calcium.
