Strain TW1: knockout of the ccpA homolog via insertion of a kanamycin cassette
Biomaterial provider
Dr. Marcelle Nascimento
Growth protocol
Growth Media: Tryptone-Vitamin broth with 0.5% galactose added OD600: 0.5
Extracted molecule
total RNA
Extraction protocol
RNA from S. mutans was isolated from 50 ml cultures that were grown under the desired conditions and harvested by centrifugation at 4ºC. Pelleted cells were resuspended in 400 ml of DEPC-treated water, 800 ml of RNA protect reagent (QIAGEN, Inc., Chatsworth, CA) was added, and the samples were incubated at room temperature for 5 min with vortexing for 10 s at one minute intervals. Cells were then pelleted, resuspended in 500 ml Tris-EDTA (50:10) buffer and transferred to 1.5 ml screw cap tubes containing sterile glass beads (0.1 mm avg. diam.; Biospec, Bartlesville, OK), 100 ml of 1% SDS and 650 ml of acid phenol:chloroform (5:1). The mixture was homogenized in a Bead Beater twice for 40 s and chilled on ice for 2 min between cycles. Samples were centrifuged for 30 min at maximum speed at 4ºC. Two additional hot acid phenol:chloroform extractions were performed followed by one extraction with chloroform:isoamyl-alcohol (24:1). RNA was precipitated in the presence of isopropanol and sodium acetate, pH 5, at -20ºC for 1 h, followed by multiple washes with 70% ethanol, one wash with 99% ethanol, and drying in vacuo. RNA was resuspended in DEPC-treated water and digested with DNAseI (Ambion, Austin, TX). The RNA was re-purified and treated on-column with DNaseI using the RNeasy mini kit (QIAGEN, Inc., Chatsworth, CA), as recommended by the supplier.
Label
Cy3
Label protocol
All RNAs were purified as described above and used to generate cDNA according to the protocol provided by TIGR with the following minor modifications. The amount of RNA in each reaction was increased to 10 ug and the molar ratio of dTTP:aa-dUTP was increased to 1:2. SuperscriptIII Reverse transcriptase (Invitrogen, Gaithersburg, MD) was used to increase cDNA yields. Purified cDNAs were coupled with indocarbocyanine (Cy3)-dUTP
RNA from S. mutans was isolated from 50 ml cultures that were grown under the desired conditions and harvested by centrifugation at 4ºC. Pelleted cells were resuspended in 400 ml of DEPC-treated water, 800 ml of RNA protect reagent (QIAGEN, Inc., Chatsworth, CA) was added, and the samples were incubated at room temperature for 5 min with vortexing for 10 s at one minute intervals. Cells were then pelleted, resuspended in 500 ml Tris-EDTA (50:10) buffer and transferred to 1.5 ml screw cap tubes containing sterile glass beads (0.1 mm avg. diam.; Biospec, Bartlesville, OK), 100 ml of 1% SDS and 650 ml of acid phenol:chloroform (5:1). The mixture was homogenized in a Bead Beater twice for 40 s and chilled on ice for 2 min between cycles. Samples were centrifuged for 30 min at maximum speed at 4ºC. Two additional hot acid phenol:chloroform extractions were performed followed by one extraction with chloroform:isoamyl-alcohol (24:1). RNA was precipitated in the presence of isopropanol and sodium acetate, pH 5, at -20ºC for 1 h, followed by multiple washes with 70% ethanol, one wash with 99% ethanol, and drying in vacuo. RNA was resuspended in DEPC-treated water and digested with DNAseI (Ambion, Austin, TX). The RNA was re-purified and treated on-column with DNaseI using the RNeasy mini kit (QIAGEN, Inc., Chatsworth, CA), as recommended by the supplier.
Label
Cy5
Label protocol
All RNAs were purified as described above and used to generate cDNA according to the protocol provided by TIGR with the following minor modifications. The amount of RNA in each reaction was increased to 10 ug and the molar ratio of dTTP:aa-dUTP was increased to 1:2. SuperscriptIII Reverse transcriptase (Invitrogen, Gaithersburg, MD) was used to increase cDNA yields. Reference cDNA was coupled with indodicarbocyanine (Cy5)-dUTP (Amersham Biosciences, Piscataway, NJ).
Hybridization protocol
Hybridizations were carried out in a Maui 4-chamber hybridization system (BioMicro Systems, Salt Lake City, Utah) for 17 h at 42ºC. The slides were then washed according to TIGR protocols (SOT# M008, version 2.1, 11/06).
Scan protocol
The slides were scanned using a GenePix Scanner (Axon Instruments Inc., Union City, CA).
Description
Streptococcus mutans ccpA mutant
Data processing
After the slides were scanned, single-channel images were loaded into TIGR Spotfinder software and overlayed. A spot grid was created according to TIGR specifications and manually adjusted to fit all spots within the grid, then the intensity values of each spot were measured. Data was normalized using Microarray data analysis software (MIDAS), by using LOWESS and Iterative Log Mean Centering with default settings.
